Pyrrolidine derivatives as oxytocin antagonists

ABSTRACT

The present invention relates to novel pyrrolidine derivative of formula (I), its geometrical isomers, its optically active forms as enantiomers, diastereomers, mixtures of these and its racemate forms, as well as salts thereof, wherein R 1  is selected from the group comprising or consisting of H and C 1 –C 6 -alkyl, for the prevention and/or treatment of preterm labor, premature birth or dysmenorrhea

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a national stage application of International PatentApplication No. PCT/EP03/50286, filed on Jul. 4, 2003, and claimspriority to European Patent Application No. 02100784.4, filed on Jun. 5,2002, both of which are incorporated herein by reference in theirentireties.

FIELD OF THE INVENTION

The present invention is directed to novel pyrrolidine derivatives, inparticular for use as medicaments, as well as pharmaceuticalformulations containing such pyrrolidine derivatives. Said pyrrolidinederivatives are useful in the treatment and/or prevention of pretermlabor, premature birth, dysmenorrhea. Preferably, the pyrrolidinederivatives display a modulatory, notably an antagonist activity of theoxytocin receptor. More preferably, said compounds are useful in thetreatment and/or prevention of disease states mediated by oxytocin,including preterm labor, premature birth and dysmenorrhea.

BACKGROUND OF THE INVENTION

Oxytocin (OT) is a cyclic nona-peptide whose actions are mediated byactivation of specific G protein-coupled receptors currently classifiedinto OT receptors (OT-R) (1).

Oxytocin (OT) causes the contraction of the uterus of mammals duringlabor. The corresponding oxytocin receptor belongs to the family ofG-protein-coupled receptors and is similar to V₁ and V₂ vasopressinreceptors. OT receptors increase dramatically during the course ofpregnancy. The concentration of OT receptors has been shown to correlatewith spontaneous uterine activity (2–3). OT-induced contractions of theuterus during labor result in the dilatation of the cervix andeventually in the movement of the foetus through the vaginal canal. Insome cases, these contractions occur before the foetus is fully viable,resulting in premature labor. Premature labor and premature birth areundesired as they are major causes of perinatal morbidity. Hence, themanagement of preterm labor represents a significant problem in thefield of obstetrics.

In recent years, strong evidence has accumulated indicating that thehormone oxytocin plays a major role in initiating labor in mammals, inparticular in humans. Thereby, it is assumed that oxytocin exerts saideffect in a direct as well as an indirect way, by contracting theuterine myometrium and by enhancing the synthesis and release ofcontractile prostaglandins from the uterine endometrium/decidua. Theseprostaglandins may furthermore play a role in the cervical ripeningprocess. This “up-regulation” of oxytocin receptors and increaseduterine sensitivity seems to be due to trophic effects of rising plasmalevels of estrogen towards term. By down-regulating oxytocin, it isexpected that both the direct (contractile) and indirect (increasedprostaglandin synthesis) effects of oxytocin on the uterus could beblocked. An oxytocin modulator, e.g. blocker or antagonist would likelybe efficacious for treating preterm labor.

A further condition related to oxytocin is dysmenorrhea, which ischaracterised by pain or discomfort associated with menses. The pain isbelieved to result from uterine contractions and ischemia, probablymediated by the effect of prostaglandins produced in the secretoryendometrium. By blocking both the indirect and direct effects ofoxytocin on the uterus, an oxytocin antagonist would be a likelycandidate for treating dysmenorrhea.

Some agents counteracting the action of oxytocin are currently used inclinical studies (4). Such tocolytic agents (i.e. uterine-relaxingagents) include beta-2-adrenergic agonists, magnesium sulfate andethanol. The leading beta-2-adrenergic agonist is Ritodrine, whichcauses a number of cardiovascular and metabolic side effects, includingtachycardia, increased renin secretion, hyperglycemia and reactivehypoglycemia in the infant. Further beta-2-adrenergic agonists,including terbutaline and albuterol have side effects similar to thoseof ritodrine. Magnesium sulfate at plasma concentrations above thetherapeutic range of 4 to 8 mg/dL can cause inhibition of cardiacconduction and neuromuscular transmiss-ion, respiratory depression andcardiac arrest, thus making this agent unsuitable when renal function isimpaired. Ethanol is as effective as ritodrine in preventing prematurelabor, but it does not produce a corresponding reduction in theincidence of fetal respiratory distress that administration of ritodrinedoes.

Atosiban, a peptide OT antagonist, suffers the problem of most peptides:low oral bioavailability resulting from intestinal degradation. Suchcompounds must be administered parenterally.

The development of non-peptide ligands for peptide hormone receptors isexpected to overcome this problem. Small molecule selective oxytocinantagonists have been reported by Merck. In addition to cyclichexapeptides, Merck suggested indanylpiperidines and tolylpiperazines asorally deliverable OT antagonists (5). In WO 96/22775 and U.S. Pat. No.5,756,497, Merck reported benzoxazinylpiperidines or benzoxazinones asOT receptor antagonists.

Specific sulfonamides have been reported to antagonize ocytocin at theocytocin receptor. Elf Sanofi's EP-A-0469984 and EP-A-0526348 reportN-sulfonyl indolines acting as antagonists of the vasopressin and theoxytocin receptors.

American Cyanamid's U.S. Pat. No. 5,889,001 claims pyrazolebenzodiazepine derivatives as vasopressin and oxytocin antagonists.

Recent pyrrolidine derivatives, such as pyrrolidine amides andpyrrolidines substituted with fused heteroaryl were developed asoxytocin receptor antagonists (WO 01/72705).

SUMMARY OF THE INVENTION

In a first aspect, the invention provides novel pyrrolidine derivativesof formula I:

R¹ in formula (I) is selected from the group consisting of H andsubstituted or unsubstituted C₁–C₆-alkyl. Preferably R¹ is H or methyl.

R² in formula (I) is selected from the group consisting of hydrogen,substituted or unsubstituted C₁–C₆-alkyl, substituted or unsubstitutedC₁–C₆-alkyl aryl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted C₁–C₆-alkyl heteroaryl, substituted or unsubstitutedC₂–C₆-alkenyl, substituted or unsubstituted C₂–C₆-alkenyl aryl,substituted or unsubstituted C₂–C₆-alkenyl heteroaryl, substituted orunsubstituted C₂–C₆-alkynyl, substituted or unsubstituted C₂–C₆-alkynylaryl, substituted or unsubstituted C₂–C₆-alkynyl heteroaryl, substitutedor unsubstituted C₃–C₈-cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted C₁–C₆-alkyl cycloalkyl,substituted or unsubstituted C₁–C₆-alkyl heterocycloalkyl, substitutedor unsubstituted C₁–C₆-alkyl carboxy, acyl, substituted or unsubstitutedC₁–C₆-alkyl acyl, substituted or unsubstituted C₁–C₆-alkyl acyloxy,substituted or unsubstituted C₁–C₆-alkyl alkoxy, alkoxycarbonyl,substituted or unsubstituted C₁–C₆-alkyl alkoxycarbonyl, aminocarbonyl,substituted or unsubstituted C₁–C₆-alkyl aminocarbonyl, substituted orunsubstituted C₁–C₆-alkyl acylamino, substituted or unsubstitutedC₁–C₆-alkyl ureido, substituted or unsubsti-tuted C₁–C₆-alkyl amino,substituted or unsubstituted C₁–C₆-alkyl sulfonyloxy, sulfonyl,substituted or unsubstituted C₁–C₆-alkyl sulfonyl; sulfinyl, substitutedor unsubstituted C₁–C₆-alkyl sulfinyl, substituted or unsubstitutedC₁–C₆-alkyl sulfanyl and substituted or unsubstituted C₁–C₆-alkylsulfonylamino.

R³ in formula (I) is selected from the group consisting of substitutedor unsubstituted aryl and substituted and unsubstituted heteroaryl.

X in formula (I) is selected from the group consisting of O or NR⁴.Thereby, R⁴ is selected from the group consisting of H, substituted orunsubstituted C₁–C₆-alkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroryl, substituted or unsubstitutedC₁–C₆-alkyl aryl, substituted and unsubstituted C₁–C₆-alkyl heteroaryl.Preferably, R⁴ is H or C₁–C₆-alkyl, like a methyl or ethyl group.

Alternatively, R² and R⁴ in formula (I) may form—together with the Natom to which they are linked—a substituted or unsubstituted, 5–8membered saturated or unsaturated heterocycloalkyl ring, e.g. apiperidinyl or piperazinyl moiety, which may be optionally fused with anaryl, heteroaryl, cycloalkyl or heterocycloalkyl ring.

n in formula (I) is an integer from 1 to 3, more preferred is 1 or 2.

In a second aspect, the present invention provides novel pyrrolidinederivatives of formula I for use as a medicament.

In a third aspect, the invention provides a pharmaceutical compositioncomprising a compound of formula I, together with a pharmaceuticallyacceptable excipient or carrier.

In a fourth aspect, the invention provides a compound of formula I, forthe preparation of a pharmaceutical composition useful in the treatmentand/or prevention of preterm labor, premature birth, dysmenorrhea.

In a fifth aspect, the invention provides a compound according toformula I for the modulation of the function of OT receptor.

In a sixth aspect, the invention provides a use of a compound of formulaI for the treatment of a disease associated with the OT receptor such aspreterm labor, premature birth, dysmenorrhea.

In a seventh aspect, the invention provides a method of treating adisease associated with the OT receptor such as preterm labor, prematurebirth, dysmenorrhea, comprising administering to a patient in needthereof an effective amount of a compound of formula I.

In an eighth aspect, the invention provides a method of synthesis of acompound according to formula I.

DETAILED DESCRIPTION OF THE INVENTION

It is an object of the present invention to provide substances which aresuitable for the treatment and/or prevention of preterm labor, prematurebirth and dysmenorrhea.

It is notably an object of the present invention to provide chemicalcompounds which are able to down-regulate, including to antagonize, thefunction of OT in disease states in mammals, especially in humans.

It is also an object of the present invention to provide small moleculechemical compounds for the modulation, preferably the down-regulation orantagonization of the oxytocin receptor.

Moreover, it is an object of the present invention to provide methodsfor preparing said small molecule chemical compounds. It is furthermorean object of the present invention to provide a new category ofpharmaceutical formulations for the treatment of preterm labor anddysmenorrhea, and/or diseases mediated by the oxytocin receptor.

It is finally an object of the present invention to provide a method forthe treatment and/or prevention of disorders mediated by the oxytocinreceptor, like preterm labor with oxytocin antagonists, acting forexample by antagonizing the binding of oxytocin to its receptor.

The following paragraphs provide definitions of the various chemicalmoieties that make up the compounds according to the invention and areintended to apply uniformly throughout the specification and claimsunless an otherwise expressly set out definition provides a broaderdefinition.

“C₁–C₆-alkyl” refers to monovalent alkyl groups having 1 to 6 carbonatoms. This term is exemplified by groups such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl and thelike.

“Aryl” refers to an unsaturated aromatic carbocyclic group of from 6 to14 carbon atoms having a single ring (e.g., phenyl) or multiplecondensed rings (e.g., naphthyl). Preferred aryl include phenyl,naphthyl, phenantrenyl and the like.

“C₁–C₆-alkyl aryl” refers to C₁–C₆-alkyl groups having an arylsubstituent, including benzyl, phenethyl and the like.

“Heteroaryl” refers to a monocyclic heteroaromatic, or a bicyclic or atricyclic fused-ring heteroaromatic group. Particular examples ofheteroaromatic groups include optionally substituted pyridyl, pyrrolyl,furyl, thienyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl,isothiazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl,1,2,3-oxadiazolyl, 1,2,4-oxadia-zolyl, 1,2,5-oxadiazolyl,1,3,4-oxadiazolyl, 1,3,4-triazinyl, 1,2,3-triazinyl, benzofuryl,[2,3-dihydro]benzofuryl, isobenzofuryl, benzothienyl, benzotriazolyl,isobenzothienyl, indolyl, isoindolyl, 3H-indolyl, benzimidazolyl,imidazo[1,2-a]pyridyl, benzothiazolyl, benzoxazolyl, quinolizinyl,quinazolinyl, pthalazinyl, quinoxalinyl, cinnolinyl, napthyridinyl,pyrido[3,4-b]pyridyl, pyrido[3,2-b]pyridyl, pyrido[4,3-b]pyridyl,quinolyl, isoquinolyl, tetrazolyl, 5,6,7,8-tetrahydroquinolyl,5,6,7,8-tetrahydroisoquinolyl, purinyl, pteridinyl, carbazolyl,xanthenyl or benzoquinolyl.

“C₁–C₆-alkyl heteroaryl” refers to C₁–C₆-alkyl groups having aheteroaryl substituent, including 2-furylmethyl, 2-thienylmethyl,2-(1H-indol-3-yl)ethyl and the like.

“C₂–C₆-alkenyl” refers to alkenyl groups preferably having from 2 to 6carbon atoms and having at least 1 or 2 sites of alkenyl unsaturation.Preferable alkenyl groups include ethenyl (—CH═CH₂), n-2-propenyl(allyl, —CH₂CH═CH₂) and the like.

“C₂–C₆-alkenyl aryl” refers to C₂–C₆-alkenyl groups having an arylsubstituent, including 2-phenylvinyl and the like.

“C₂–C₆-alkenyl heteroaryl” refers to C₂–C₆-alkenyl groups having aheteroaryl substituent, including 2-(3-pyridinyl)vinyl and the like.

“C₂–C₆-alkynyl” refers to alkynyl groups preferably having from 2 to 6carbon atoms and having at least 1–2 sites of alkynyl unsaturation,preferred alkynyl groups include ethynyl (—C≡CH), propargyl (—CH₂C≡CH),and the like.

“C₂–C₆-alkynyl aryl” refers to C₂–C₆-alkynyl groups having an arylsubstituent, including phenylethynyl and the like.

“C₂–C₆-alkynyl heteroaryl” refers to C₂–C₆-alkynyl groups having aheteroaryl substituent, including 2-thienylethynyl and the like.

“C₃–C₈-cycloalkyl” refers to a saturated carbocyclic group of from 3 to8 carbon atoms having a single ring (e.g., cyclohexyl) or multiplecondensed rings (e.g., norborn yl). Preferred cycloalkyl includecyclopentyl, cyclohexyl, norbornyl and the like.

“Heterocycloalkyl” refers to a C₃–C₈-cycloalkyl group according to thedefinition above, in which up to 3 carbon atoms are replaced byheteroatoms chosen from the group consisting of O, S, NR, R beingdefined as hydrogen or methyl. Preferred heterocycloalkyl includepyrrolidine, piperidine, piperazine, 1-methylpiperazine, morpholine, andthe like.

“C₁–C₆-alkyl cycloalkyl” refers to C₁–C₆-alkyl groups having acycloalkyl substituent, including cyclohexylmethyl, cyclopentylpropyl,and the like.

“C₁–C₆-alkyl heterocycloalkyl” refers to C₁–C₆-alkyl groups having aheterocycloalkyl substituent, including 2-(1-pyrrolidinyl)ethyl,4-morpholinylmethyl, (1-methyl-4-piperidinyl)methyl and the like.

“Carboxy” refers to the group —C(O)OH.

“C₁–C₆-alkyl carboxy” refers to C₁–C₅-alkyl groups having an carboxysubstituent, including 2-carboxyethyl and the like.

“Acyl” refers to the group —C(O)R where R includes “C₁–C₆-alkyl”,“aryl”, “heteroaryl”, “C₁–C₆-alkyl aryl” or “C₁–C₆-alkyl heteroaryl”.

“C₁–C₆-alkyl acyl” refers to C₁–C₆-alkyl groups having an acylsubstituent, including 2-acetylethyl and the like.

“Acyloxy” refers to the group —OC(O)R where R includes “C₁–C₆-alkyl”,“aryl”, “heteroaryl”, “C₁–C₆-alkyl aryl” or “C₁–C₆-alkyl heteroaryl”.

“C₁–C₆-alkyl acyloxy” refers to C₁–C₆-alkyl groups having an acyloxysubstituent, including 2-(acetyloxy)ethyl and the like.

“Alkoxy” refers to the group —O—R where R includes “C₁–C₆-alkyl” or“aryl” or “heteroaryl” or “C₁–C₆-alkyl aryl” or “C₁–C₆-alkylheteroaryl”. Preferred alkoxy groups include by way of example, methoxy,ethoxy, phenoxy and the like.

“C₁–C₆-alkyl alkoxy” refers to C₁–C₅-alkyl groups having an alkoxysubstituent, including 2-ethoxyethyl and the like.

“Alkoxycarbonyl” refers to the group —C(O)OR where R includes H,“C₁–C₆-alkyl” or “aryl” or “heteroaryl” or “C₁–C₆-alkyl aryl” or“C₁–C₆-alkyl heteroaryl”.

“C₁–C₆-alkyl alkoxycarbonyl” refers to C₁–C₆-alkyl groups having analkoxycarbonyl substituent, including 2-(benzyloxycarbonyl)ethyl and thelike.

“Aminocarbonyl” refers to the group —C(O)NRR′ where each R, R′ includesindependently hydrogen or C₁–C₆-alkyl or aryl or heteroaryl or“C₁–C₆-alkyl aryl” or “C₁–C₆-alkyl heteroaryl”.

“C₁–C₆-alkyl aminocarbonyl” refers to C₁–C₆-alkyl groups having anaminocarbonyl substituent, including 2-(dimethylaminocarbonyl)ethyl andthe like.

“Acylamino” refers to the group —NRC(O)R′ where each R, R′ isindependently hydrogen or “C₁–C₆-alkyl” or “aryl” or “heteroaryl” or“C₁–C₆-alkyl aryl” or “C₁–C₆-alkyl heteroaryl”.

“C₁–C₆-alkyl acylamino” refers to C₁–C₆-alkyl groups having an acylaminosubstituent, including 2-(propionylamino)ethyl and the like.

“Ureido” refers to the group —NRC(O)NR′R″ where each R, R′, R″ isindependently hydrogen, “C₁–C₆-alkyl”, “C₂–C₆-alkenyl”, “C₂–C₆-alkynyl”,“C₃–C₈-cycloalkyl”, “heterocycloalkyl”, “aryl”, “heteroaryl”,“C₁–C₆-alkyl aryl” or “C₁–C₆-alkyl heteroaryl”, “C₂–C₆-alkenyl aryl”,“C₂–C₆-alkenyl heteroaryl”, “C₂–C₆-alkynyl aryl”,“C₂–C₆-alkynylheteroaryl”, “C₁–C₆-alkyl cycloalkyl”, “C₁–C₆-alkylheterocycloalkyl”, and where R′ and R″, together with the nitrogen atomto which they are attached, can optionally form a 3–8-memberedheterocycloalkyl ring.

“C₁–C₆-alkyl ureido” refers to C₁–C₆-alkyl groups having an ureidosubstituent, including 2-(N′-methylureido)ethyl and the like.

“Carbamate” refers to the group —NRC(O)OR′ where each R, R′ isindependently hydrogen, “C₁–C₆-alkyl”, “C₂–C₆-alkenyl”, “C₂–C₆-alkynyl”,“C₃–C₈-cycloalkyl”, “heterocycloalkyl”, “aryl”, “heteroaryl”,“C₁–C₆-alkyl aryl” or “C₁–C₆-alkyl heteroaryl”, “C₂–C₆-alkenyl aryl”,“C₂–C₆-alkenyl heteroaryl”, “C₂–C₆-alkynyl aryl”,“C₂–C₆-alkynylheteroaryl”, “C₁–C₆-alkyl cycloalkyl”, “C₁–C₆-alkylheterocycloalkyl”.

“Amino” refers to the group —NRR′ where each R,R′ is independentlyhydrogen or “C₁–C₆-alkyl” or “aryl” or “heteroaryl” or “C₁–C₆-alkylaryl” or “C₁–C₆-alkyl heteroaryl”, or “cycloalkyl”, or“heterocycloalkyl”, and where R and R′, together with the nitrogen atomto which they are attached, can optionally form a 3–8-memberedheterocycloalkyl ring.

“C₁–C₆-alkyl amino” refers to C₁–C₅-alkyl groups having an aminosubstituent, including 2-(1-pyrrolidinyl)ethyl and the like.

“Ammonium” refers to a positively charged group —N⁺RR′R″, where eachR,R′,R″ is independently “C₁–C₆-alkyl” or “C₁–C₆-alkyl aryl” or“C₁–C₆-alkyl heteroaryl”, or “cycloalkyl”, or “heterocycloalkyl”, andwhere R and R′, together with the nitrogen atom to which they areattached, can optionally form a 3–8-membered heterocycloalkyl ring.

“Halogen” refers to fluoro, chloro, bromo and iodo atoms.

“Sulfonyloxy” refers to a group —OSO₂—R wherein R is selected from H,“C₁–C₆-alkyl”, “C₁–C₆-alkyl” substituted with halogens, e.g., an—OSO₂—CF₃ group, “aryl”, “heteroaryl”, “(C₁–C₆-alkyl aryl” or“C₁–C₆-alkyl heteroaryl”.

“C₁–C₆-alkyl sulfonyloxy” refers to C₁–C₆-alkyl groups having asulfonyloxy substituent, including 2-(methylsulfonyloxy)ethyl and thelike.

“Sulfonyl” refers to group “—SO₂—R” wherein R is selected from H,“aryl”, “heteroaryl”, “C₁–C₆-alkyl”, “C₁–C₆-alkyl” substituted withhalogens, e.g., an —SO₂—CF₃ group, “C₁–C₆-alkyl aryl” or “C₁–C₆-alkylheteroaryl”.

“C₁–C₆-alkyl sulfonyl” refers to C₁–C₆-alkyl groups having a sulfonylsubstituent, including 2-(methylsulfonyl)ethyl and the like.

“Sulfinyl” refers to a group “—S(O)—R” wherein R is selected from H,“C₁–C₆-alkyl”, “C₁–C₆-alkyl” substituted with halogens, e.g., an —SO—CF₃group, “aryl”, “heteroaryl”, “C₁–C₆-alkyl aryl” or “C₁–C₆-alkylheteroaryl”.

“C₁–C₆-alkyl sulfinyl” refers to C₁–C₆-alkyl groups having a sulfinylsubstituent, including 2-(methylsulfinyl)ethyl and the like.

“Sulfanyl” refers to groups —S—R where R includes “C₁–C₆-alkyl” or“aryl” or “hetero-aryl” or “C₁–C₆-alkyl aryl” or “C₁–C₆-alkylheteroaryl”. Preferred sulfanyl groups include methylsulfanyl,ethylsulfanyl, and the like.

“C₁–C₆-alkyl sulfanyl” refers to C₁–C₆-alkyl groups having a sulfanylsubstituent, including 2-(ethylsulfanyl)ethyl and the like.

“Sulfonylamino” refers to a group —NRSO₂—R′ where each R, R′ isindependently hydrogen or “C₁–C₆-alkyl” or “aryl” or “heteroaryl” or“C₁–C₆-alkyl aryl” or “C₁–C₆-alkyl heteroaryl”.

“C₁–C₆-alkyl sulfonylamino” refers to C₁–C₆-alkyl groups having asulfonylamino substituent, including 2-(ethylsulfonylamino)ethyl and thelike.

“Substituted or unsubstituted”: Unless otherwise constrained by thedefinition of the individual substituent, the above set out groups, like“alkyl”, “alkenyl”, “alkynyl”, “aryl” and “heteroaryl” etc. groups canoptionally be substituted with from 1 to 5 substituents selected fromthe group consisting of “C₁–C₆-alkyl”, “C₂–C₆-alkenyl”, “C₂–C₆-alkynyl”,“cycloalkyl”, “heterocycloalkyl”, “C₁–C₆-alkyl aryl”, “C₁–C₆-alkylheteroaryl”, “C₁–C₆-alkyl cycloalkyl”, “C₁–C₆-alkyl heterocycloalkyl”,“amino”, “ammonium”, “acyl”, “acyloxy”, “acylamino”, “aminocarbonyl”,“alkoxycarbonyl”, “ureido”, “carbamate,” “aryl”, “heteroaryl”,“sulfinyl”, “sulfonyl”, “alkoxy”, “sulfanyl”, “halogen”, “carboxy”,trihalomethyl, cyano, hydroxy, mercapto, nitro, and the like.Alternatively said substitution could also comprise situations whereneighbouring substituents have undergone ring closure, notably whenvicinal functional substituents are involved, thus forming, e.g.,lactams, lactons, cyclic anhydrides, but also acetals, thioacetals,aminals formed by ring closure for instance in an effort to obtain aprotective group.

“Pharmaceutically acceptable salts or complexes” refers to salts orcomplexes of the below-specified compounds of formula (I). Examples ofsuch salts include, but are not restricted, to base addition saltsformed by reaction of compounds of formula (I) with organic or inorganicbases such as hydroxide, carbonate or bicarbonate of a metal cation suchas those selected in the group consisting of alkali metals (sodium,potassium or lithium), alkaline earth metals (e.g. calcium ormagnesium), or with an organic primary, secondary or tertiary alkylamine. Amine salts derived from methylamine, dimethylamine,trimethylamine, ethylamine, diethylamine, triethylamine, morpholine,N-Me-D-glucamine, N,N′-bis(phenylmethyl)-1,2-ethanediamine,tromethamine, ethanolamine, diethanolamine, ethylenediamine,N-methylmorpholine, procaine, piperidine, piperazine and the like arecontemplated being within the scope of the instant invention.

Also comprised are salts which are formed from to acid addition saltsformed with inorganic acids (e.g. hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid, and the like), as well assalts formed with organic acids such as acetic acid, oxalic acid,tartaric acid, succinic acid, malic acid, fumaric acid, maleic acid,ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid,polyglutamic acid, naphthalene sulfonic acid, naphthalene disulfonicacid, and poly-galacturonic acid.

“Pharmaceutically active derivative” refers to any compound that uponadministration to the recipient, is capable of providing directly orindirectly, the activity disclosed herein.

“Enantiomeric excess” (ee) refers to the products that are obtained byan asymmetric synthesis, i.e. a synthesis involving non-racemic startingmaterials and/or reagents or a synthesis comprising at least oneenantioselective step. “ee” is the percentage of excess of the majorenantiomer vs minor enantiomer [% ee=% major−% minor]. In the absence ofan asymmetric synthesis, racemic products are usually obtained that dohowever also have an activity as OT-R antagonists.

The term “preterm labor” or the term “premature labor” shall meanexpulsion from the uterus of an infant before the normal end ofgestation, or more particularly, onset of labor with effacement anddilation of the cervix before the 37^(th) week of gestation. It may ormay not be associated with vaginal bleeding or rupture of the membranes.

The term “dysmenorrhea” shall mean painful menstruation.

The term “caesarean delivery” shall mean incision through the abdominaland uterine walls for delivery of a foetus.

The present invention also includes the geometrical isomers, theoptically active forms, enantiomers, diastereomers of compoundsaccording to formula I, mixtures of these, racemates and alsopharmaceutically acceptable salts.

The compounds according to the present invention are those of formula I.

R¹ in formula (I) is selected from the group consisting of H andsubstituted or unsubstituted C₁–C₆-alkyl. Preferably R¹ is H or methyl.

R² in formula (I) is selected from the group consisting of hydrogen,substituted or unsubstituted C₁–C₆-alkyl, substituted or unsubstitutedC₁–C₆-alkyl aryl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted C₁–C₆-alkyl heteroaryl, substituted or unsubstitutedC₂–C₆-alkenyl, substituted or unsubstituted C₂–C₆-alkenyl aryl,substituted or unsubstituted C₂–C₆-alkenyl heteroaryl, substituted orunsubstituted C₂–C₆-alkynyl, substituted or unsubstituted C₂–C₆-alkynylaryl, substituted or unsubstituted C₂–C₆-alkynyl heteroaryl, substitutedor unsubstituted C₃–C₈-cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted C₁–C₆-alkyl cycloalkyl,substituted or unsubstituted C₁–C₆-alkyl heterocycloalkyl, substitutedor unsubstituted C₁–C₆-alkyl carboxy, acyl, substituted or unsubstitutedC₁–C₆-alkyl acyl, substituted or unsubstituted C₁–C₆-alkyl acyloxy,substituted or unsubstituted C₁–C₆-alkyl alkoxy, alkoxycarbonyl,substituted or unsubstituted C₁–C₆-alkyl alkoxycarbonyl, aminocarbonyl,substituted or unsubstituted C₁–C₆-alkyl aminocarbonyl, substituted orunsubstituted C₁–C₆-alkyl acylamino, substituted or unsubstitutedC₁–C₆-alkyl ureido, substituted or unsubsti-tuted C₁–C₆-alkyl amino,substituted or unsubstituted C₁–C₆-alkyl sulfonyloxy, sulfonyl,substituted or unsubstituted C₁–C₆-alkyl sulfonyl, sulfinyl, substitutedor unsubstituted C₁–C₆-alkyl sulfinyl, substituted or unsubstitutedC₁–C₆-alkyl sulfanyl, substituted and unsubstituted C₁–C₆-alkylsulfonylamino.

R³ in formula (I) is selected from the group consisting of substitutedor unsubstituted aryl and substituted or unsubstituted heteroaryl.

X in formula (I) is selected from the group consisting of O or NR⁴,wherein R⁴ is selected from the group consisting of H, substituted orunsubstituted C₁–C₆-alkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroryl, substituted or unsubstitutedC₁–C₆-alkyl aryl and substituted or unsubstituted C₁–C₆-alkylheteroaryl. Preferably, R⁴ is H or C₁–C₆-alkyl, like a methyl or ethylgroup.

Alternatively, R² and R⁴ in formula (I) may form—together with the Natom to which they are linked—a substituted or unsubstituted, 5–8membered saturated or unsaturated heterocycloalkyl ring, e.g. apiperidinyl or piperazinyl moiety, which may be optionally fused with anaryl, heteroaryl, cycloalkyl or heterocycloalkyl ring.

n in formula (I) is an integer from 1 to 3, more preferred is 1 or 2.

Preferred R² in compounds according to formula I are those that areselected from the group consisting of H, acyl, preferably an acetylmoiety, an aryl, optionally substituted by a substituted orunsubstituted C₁–C₆-alkoxy, e.g. a methyloxy-phenyl goup, a C₁–C₃ alkyllike methyl or ethyl, optionally substituted by an substituted orunsubstituted acyl group or ester group, preferably formic acid oracetic acid t-butyl ester, N-(2-pyrrolidin-1-yl-ethyl)acetamide, andoptionally substituted by an substituted or unsubstituted heteroaryl,preferably N-pyrazole.

Preferred R³ in compounds according to formula I are those that areselected from the group consisting of aryl group optionally substitutedby a substituted or unsubstituted aryl group. Particularly preferred R³is a biphenyl or 2-methyl biphenyl moiety.

A particularly preferred embodiment of the present invention is apyrrolidine derivative according to formula I wherein X is O or NH and nis 1 or 2.

Another preferred embodiment of the present invention is a pyrrolidinederivative according to formula I wherein X is NR⁴ and wherein R⁴ and R²form a saturated or unsaturated, substituted or unsubstituted, fused orunfused, heterocyclic ring with the N atom they are linked to,preferably a 5 or 6-membered ring, more preferably a piperidine, amethylpiperazine or an isoindole-1,3-dione.

Compounds of formula I may be used for the treatment of a disease.

Specifically, the compounds of formula I are suitable for use intreating disorders such as preterm labor, premature birth, dysmenorrheaand for stopping labor prior to cesarean delivery. The compounds of thepresent invention are in particular useful for the treatment of pretermlabor, premature birth and dysmenorrhea.

Preferably, the compounds according to Formula I alone or in a form of apharmaceutical composition are suitable for the modulation of oxytocinfunction(s), thus specifically allowing the treatment and/or preventionof disorders which are mediated by the oxytocin receptor. Suchmodulation preferably involves the inhibition of OT-R function(s),notably by the antagonization of the oxytocin receptor in mammals, andin particular in humans.

Abnormal activity or hyperactivity of the oxytocin receptor arefrequently involved in various disorders including the above enumerateddisorders and disease states. Hence, the compounds according to theinvention may be used for the treatment of disorders by modulating OT-Rfunction or pathways. The modulation of the OT-R function or pathwaysmay involve the down-regulation and/or inhibition of the oxytocinreceptor. The compounds of the invention may be employed alone or incombination with further pharmaceutical agents, e.g. with a further OT-Rmodulator.

When employed as pharmaceuticals, the pyrrolidine derivatives of thepresent invention are typically administered in the form of apharmaceutical composition. Hence, pharmaceutical compositionscomprising a compound of Formula I and a pharmaceutically acceptablecarrier, diluent or excipient are also within the scope of the presentinvention. A person skilled in the art is aware of a whole variety ofsuch carriers, diluents or excipients suitable to formulate apharmaceutical composition.

The compounds of the invention, together with a conventionally employedadjuvant, car-rier, diluent or excipient may be formulated aspharmaceutical compositions and unit dosages thereof, and in such formmay be employed as solids, such as tablets or filled capsules, orliquids such as solutions, suspensions, emulsions, elixirs, or capsulesfilled with the same, all for oral use, or in the form of sterileinjectable solutions for parenteral (including subcutaneous) use. Suchpharmaceutical compositions and unit dosage forms thereof may compriseingredients in conventional proportions, with or without additionalactive compounds or principles, and such unit dosage forms may containany suitable effective amount of the active ingredient commensurate withthe intended daily dosage range to be employed.

When employed as pharmaceuticals, the pyrrolidine derivatives of thisinvention are typically administered in the form of a pharmaceuticalcomposition. Such compositions can be prepared in a manner well known inthe pharmaceutical art and comprise at least one active compound.Generally, the compounds of this invention are administered in apharmaceutically effective amount. The amount of the compound actuallyadministered will typically be determined by a physician, in the lightof the relevant circumstances, including the condition to be treated,the chosen route of administration, the actual compound administered,the age, weight, and response of the individual patient, the severity ofthe patient's symptoms, and the like.

The pharmaceutical compositions of the invention can be administered bya variety of routes including oral, rectal, transdermal, subcutaneous,intravenous, intramuscular, and intranasal. Depending on the intendedroute of delivery, the compounds are preferably formulated as eitherinjectable or oral compositions. The compositions for oraladminis-tration can take the form of bulk liquid solutions orsuspensions, or bulk powders. More commonly, however, the compositionsare presented in unit dosage forms to facilitate accurate dosing. Theterm “unit dosage forms” refers to physically discrete units suitable asunitary dosages for human subjects and other mammals, each unitcontaining a predetermined quantity of active material calculated toproduce the desired therapeutic effect, in association with a suitablepharmaceutical excipient. Typical unit dosage forms include prefilled,premeasured ampoules or syringes of the liquid compositions or pills,tablets, capsules or the like in the case of solid compositions. In suchcompositions, the pyrrolidine compound is usually a minor component(from about 0.1 to about 50% by weight or preferably from about 1 toabout 40% by weight) with the remainder being various vehicles orcarriers and processing aids helpful for forming the desired dosingform.

Liquid forms suitable for oral administration may include a suitableaqueous or nonaqueous vehicle with buffers, suspending and dispensingagents, colorants, flavors and the like. Solid forms may include, forexample, any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatine; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate; a glidant such as colloidal silicon dio-xide; asweetening agent such as sucrose or saccharin; or a flavoring agent suchas pepper-mint, methyl salicylate, or orange flavoring.

Injectable compositions are typically based upon injectable sterilesaline or phosphate-buffered saline or other injectable carriers knownin the art. As above mentioned, the pyrrolidine derivatives of Formula Iin such compositions is typically a minor component, frequently rangingbetween 0.05 to 10% by weight with the remainder being the injectablecarrier and the like.

The above described components for orally administered or injectablecompositions are merely representative. Further materials as well asprocessing techniques and the like are set out in Part 8 of (6).

The compounds of this invention can also be administered in sustainedrelease forms or from sustained release drug delivery systems. Adescription of representative sustained release materials can also befound in (6).

Still a further object of the present invention is a process forpreparing pyrrolidine derivatives according to Formula I.

The pyrrolidine derivatives exemplified in this invention may beprepared from readily available or previously described startingmaterials using the following general methods and procedures. It will beappreciated that where typical or preferred experimental conditions(i.e. reaction temperatures, time, moles of reagents, solvents, etc.)are given, other experimental conditions can also be used unlessotherwise stated. Optimum reaction conditions may vary with theparticular reactants or solvents used, but such conditions can bedetermined by one skilled in the art by routine optimisation procedures.

Synthesis of Compounds of the Invention:

Examples of synthetic pathways for compounds of formula I will bedescribed below.

The following abbreviations refer respectively to the definitions below:

-   ACN (Acetonitrile)-   Boc (t-butoxycarbonyl)-   CDCl₃ (deuterated chloroform)-   cHex (Cyclohexane)-   DCM (Dichloromethane)-   DECP (Diethylcyanophosphonate)-   DIC (Diisopropyl carbodiimide)-   DIEA (disopropylethylamine)-   DMAP (4- Dimethylaminopyridine)-   DMF (Dimethylformamide)-   DMSO (Dimethylsulfoxide)-   DMSO-d₆ (deuterated dimethylsulfoxide)-   EDC (1-(3-Dimethyl-amino-propyl)-3-ethylcarbodiimide)-   EtOAc (Ethyl acetate)-   Et₂O (Diethyl ether)-   HATU    (O-(7-azabenzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexaflurophosphonate)-   K₂CO₃ (potassium carbonate)-   MEK (methylethylketone)-   MgSO₄ (Magnesium sulfate)-   NaH (Sodium hydride)-   NaHCO₃ (Sodium bicarbonate)-   nBuLi (n Butyllithium)-   NMO (N-methylmorpholine N-oxide monohydrate)-   PetEther (Petroleum ether)-   OMs (O-mesylate=O-methylsulfonate)-   OTs (O-tosylate=O-toluenesulfonate)-   TBAF (t-butylammonium fluoride)-   TBDMS (t-butyldimethylsilyl)-   TBTU    (O-Benzotriazolyl-N,N,N′,N′-tetramethyluronium-tetrafluoroborate)-   TEA (Triethyl amine)-   TFA (Trifluoro-acetic acid)-   THF (Tetrahydrofuran)-   TPAP (tetrapropylammoniumperruthenate)-   rt (room temperature).    a) Alkoxypyrrolidines:    Introduction of the R² Moiety

Alkoxypyrrolidine derivatives according to the general formula Ia(formula I wherein X is O), wherein R¹, R², R³ and n are as defined forformula I, can be prepared from the corresponding pyrrolidinederivatives of formula II, wherein R¹, R³ and n are as defined above.Alcohol derivatives of formula II are subjected to a direct O-alkylationby using a suitable alkylating agent R²-LG, wherein LG is a suitableleaving group including Cl, Br, I or OMs, OTs. Alternatively, compoundsIa may be obtained by a Mitsunobu-type reaction as outlined in Scheme 1.

An alternative route for the synthesis of alkoxypyrrolidine derivativesaccording to the general formula Ia (formula I wherein X is O), can bethe preparation of an intermediate of general formula IIa wherein R¹,R³, n and LG are as defined above by reaction of an alcohol of formulaII with MsCl, TsCl or a halogenating agent like PPh₃Br₂. The leavinggroup LG is then displaced by R²OM whereby R² is as above defined and Mis H or a metal like Na to lead to compound of formula Ia.

Introduction of the Oxime Moiety

Compound of general formula II—whereby R¹, R³ and n are as definedabove—can be prepared from compounds of general formula IV wherein R³and n are as defined above and where PG₁ is a suitable alcoholprotecting group, preferably a TBDMS. Ketone of general formula IV isreacted with a hydroxylamine derivative of general formula V wherein R¹is as defined above. PG1 is removed via a deprotection step usingstandard synthetic techniques as shown in Scheme 2.

Hydroxylamine V, if not commercially available can, for example, besynthesized by reaction of N-Boc-hyroxylamine with the correspondingalkylating agent of formula VI whereby R¹ is as above defined andX_(a)=Cl, Br, I using standard conditions as outlined in Scheme 3.

Formation of Ketopyrrolidines:

Ketopyrrolidines of general formula IV wherein R³, n and PG₁ are asabove defined can be obtained from the corresponding hydroxy pyrrolidinederivatives of formula VII wherein R³, n and PG₁ are as above defined bytreatment with appropriate oxidating agent e.g. DMSO/(COCl)₂/TEA (Swernconditions) or TPAP in the presence of NMO as illustrated in Scheme 4.

Reduction Step

Hydroxy pyrrolidine derivatives of general formula VII—wherein R³ and nare as described above and PG₁ is a protecting group—may be obtained byreduction of the corresponding pyrrolidine carboxylic derivatives offormula VIII—wherein R³ and n are as above defined, R⁷ is H or an alkylgroup and PG₂ is a suitable protecting group—after appropriateprotection/deprotection steps as described in Scheme 5. A preferredreducing agent is LiBH₄ when R⁷ is an alkyl group or LAH or BH₃.DMS whenR⁷ is H.

Coupling Step

Protected pyrrolidines carboxylic derivatives of general formula VIIIwherein R³, n, R⁷ and PG₂ are as above defined are prepared by reactionof a compound of general formula IX—wherein n, R⁷ and PG₂ are as abovedefined and PG₃ is H or a suitable N-protecting group, preferablyBoc—with an acylating agent of general formula R³—CO—Y—wherein R³ is asdefined above and Y is any appropriate leaving group—as illustrated inScheme 6.

Preferred acylating agents are acid chlorides (Y=Cl) or carboxylic acids(Y=OH) used in conjunction with an appropriate peptide coupling agentsuch as e.g. DIC, EDC, HATU, DECP or others.

Generally, the starting materials are compounds of formula IX which canbe obtained from commercial sources (e.g. protected 3-hydroxyproline,homo-3-hydroxyproline, 3-hydroxy pyrrolidine 5-propionic acid).

Other starting materials (such as compounds of formula XV, XVI, XIX andXX) can be obtained from commercially available compounds of formula IXvia intermediates of formula XII.

In this case, first carboxylic derivatives of general formula IX can bereduced to derivatives of general formula XII whereby PG₂, PG₃, R⁷ areas above defined and n=2 or 3 as described in Scheme 7.

Then, compounds of formula XII are subjected to classicalprotection/deprotection and functional group transformations, especiallyone or two carbons homologation procedures well known by the one skilledin the art (7, 8).

One preferred process consists in the homologation by one carbon ofcompounds of general formula XII wherein PG₂ and PG₃ are as abovedefined and n is 2 or 3 by displacement of a leaving group by a cyanidefollowed either by an hydrolysis to give carboxylic acids of generalformula XV whereby PG₂, PG₃ and n are as above defined or a reduction togive amino compounds of general formula XVI whereby PG₂, PG₃ and n areas above defined as described in Scheme 8.

In case of a two-carbon homologation, one preferred procedure consistsin reacting an aldehyde of general formula XVIII obtained by oxidationof a compound of general formula XII whereby PG₂, PG₃ and n are as abovedefined with a Wittig-Horner reagent as described in Scheme 9. Thecompound thus obtained is then reduced to compounds of general formulaXIX whereby PG₂, PG₃, n and R⁷ are as above defined.

The four principal chemical transformations described above, i.e. thecoupling step, the reduction step, the oxime formation and theintroduction of the R² group can be performed in a different order. Themost appropriate choice of the synthetic sequence will depend on thenature of the substituents R¹–R⁴, n, X, and other parameters that can beappreciated by those skilled in the art.

b) Aminopyrrolidines:

Introduction of the R² Group:

Aminoalkylpyrrolidine derivatives according to the general formula Ib(formula I wherein X is NR⁴), whereby R¹–R⁴ and n are as defined informula I, can be prepared from the corresponding pyrrolidinederivatives IIa obtained in a) from compounds of formula II according toscheme 1), wherein R₁, R₃, n and LG are as above defined by displacementof the LG group with the corresponding amine HNR²R⁴, as outlined isScheme 10.

An alternative way for the preparation of aminoalkylpyrrolidinederivatives according to general formula Ib (formula I wherein X is NR⁴)wherein R¹–R⁴ and n are as defined above is described in Scheme 11.According to such process, the hydroxy moiety in the pyrrolidinederivatives of general formula II (that can be obtained from compoundsof formula IV; see scheme 2) wherein R¹, R³ and n are as above definedis oxidized into the corresponding aldehyde using well known conditionsfor such transformation, e.g. DMSO/(COCl)₂, TEA (Swern conditions) orDess Martin reagent. The aldehyde is then reacted with amines HNR²R⁴whereby R² and R⁴ are as above defined under reducing conditions.

In the case of aminoalkylpyrrolidine derivatives according to generalformula Ib wherein R⁴ is H and R¹, R², R³ and n are as defined above, analternative synthetic approach may be adopted. Aminoalkylpyrrolidinederivatives according to general formula Ib (formula I wherein X is NH)can then be obtained from the corresponding aminoalkylpyrrolidinederivatives of formula III wherein R¹, R³ and n are as defined above bydirect alkylation with R²-LG wherein R² and LG are as above defined orreductive alkylation with aldehyde of the formula R²CHO—wherein R² is asdefined above—and using an appropriate reducing agent as illustrated inScheme 12.

Aminoalkyl derivatives of formula III wherein R¹, R³ and n are as abovedefined can be obtained from hydroxyalkylpyrrolidine of formula II(which can be obtained from compounds of formula IV; see scheme 2)wherein R¹, R³ and n are as defined above or derivatives of generalformula IIa (which can be obtained from compounds of formula IIaccording to scheme 1) wherein R¹, R³, n and LG are as above defined bywell known procedures for such functional group transformations. Twoexamples of such transformations are illustrated in Scheme 13.

For compounds of formula Ib (formula I wherein X is NR⁴) wherein R² isCOR⁵, SO₂R⁵, COOR⁵, CONR⁵R⁶, SO₂NR⁵R⁶ whereby R⁵ and R⁶ are substitutedor unsubstituted alkyl or aryl group and R⁴ is H or a substituted orunsubstituted alkyl or aryl group, the methods described above in Scheme10, 11 and 12 are not applicable.

In this case these compounds of formula Ib can then be obtained bytreatment of a compound of general formula Ic (compound of formula Ibwherein R² is H obtained according to methods set out in Schemes 10, 11or 12 with a suitable acylating agent, including acylchlorid or acarboxylic acid in conjunction with a peptide coupling agent, e.g. DICor EDC, a sulfonating agent and others as outlined in Scheme 14.

The other steps, i.e. introduction of the oxime moiety, formation ofketopyrrolidines, reduction step and coupling step, have already beendescribed in the above item a).

However, like for the synthesis of alkoxypyrrolidines, the fourprincipal chemical transformations described above, can be performed ina different order. The most appropriate choice of the synthetic sequencewill depend on the nature of the substituents R¹–R⁴, n, X, and otherparameters that can be appreciated by those skilled in the art.

As an example, compounds of general formula I whereby R¹–R⁴, n and X areas above defined can be obtained from ketopyrrolidine of general formulaX whereby R², R³, n and X are as defined above by reaction with ahydroxylamine V whereby R₁ is as above defined as described in Scheme15, which leads to the introduction of the oxime moiety.

The ketopyrrolidine of formula X can be obtained by oxidation of analcohol of general formula XI whereby R², R³, n and X are as definedabove and PG₂ is H or a suitable O-protecting group under similarconditions as for the transformation of compounds of general formula VIIin compounds of general formula IV as described in Scheme 16.

Compounds of general formula XI wherein R², R³, n, PG₂ and X are asdefined above can be obtained from compounds of general formula VII,wherein R³, n and PG1 are as above defined by the introduction of the R²group following one of the processes described in Schemes 1, 10, 11, 12,13 or 14. The choice of the process will depend on the nature of R³, R²,n and X and will be appreciated by those skilled in the art as well asthe choice and sequence of appropriate protection/deprotection steps asdescribed in Scheme 17.

The obtention of compounds of formula VII, like the reduction step hasbeen already described above.

The reaction sequences outlined in the above Schemes provideenantiomerically pure compounds of formula I, if enantiomerically purestarting materials are used. (R)- as well as (S)-enantiomers can beobtained depending upon whether (R)- or (S)-forms of commerciallyavailable compounds of formulas IX were used as the starting materials.

The reaction sequences outlined in the above reaction schemes usuallyprovide mixtures of (E)- and (Z)-isomers with respect to thesubstituents on the exocyclic double bond of the pyrrolidine ring.(E)/(Z)-isomers could be separated by standard chromatography techniqueswell known to the person skilled in the art, such as by reversed phasehigh-pressure liquid chromatography (HPLC) or silica gel flashchromatography (FC). Alternatively, either one of the (E)/(Z)isomerscould successively be enriched by selective crystallisation inappropriate solvents or solvent mixtures. The assignment of the absoluteconfiguration of the exocyclic double bond was performed usingNMR-techniques well described in the literature as will be known to thepractitioner skilled in the art (for configuration assignments of e.g.oxime functionalities (9). In order to increase the overall yields ofone isomer (usually the (Z)-isomer), the other isomer (usually the(E)-isomer) could be recycled by deliberate re-isomerization in organicsolvents containing traces of acid, such as HCl, followed again by(E)/(Z)-separation through chromatography and /or crystallisation.

According to a further general process (scheme 18), compounds of formulaI can be converted to alternative compounds of formula I, employingsuitable interconversion techniques such as hereinafter described in theExamples.

If the above set out general synthetic methods are not applicable forobtaining compounds according to formula I and/or necessaryintermediates for the synthesis of compounds of formula I, suitablemethods of preparation known by a person skilled on the art should beused. In general, the synthesis pathways for any individual compound offormula I will depend on the specific substitutents of each molecule andupon the ready availability of intermediates necessary; again suchfactors being appreciated by those of ordinary skill in the art. For allthe protection, deprotection methods, see (7, 10).

EXAMPLES

The invention will be illustrated by means of the following exampleswhich are not to be construed as limiting the scope of the invention.

The compounds of the present invention may be synthesized according tothe different synthesis pathways provided above. The following examplesillustrate preferred methods for synthesizing the compounds according toformula I, and for determining their biological activities.

Example I (3EZ,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4yl)carbonyl] pyrrolidin-3-one O-methyloxime (1) (Compound of Formula II orFormula Ia wherein R² is H)

Intermediate (1a): 1-(tert-butoxycarbonyl)-4-oxo-L-proline

Ketopyrrolidine Formation:

Commercial (2S,4R)-1-(tert-butoxycarbonyl)-4-hydroxy-2-pyrrolidinecarboxylic acid (30g, 0.13 mol) was dissolved in acetone (1500 ml). A mechanical stirrerwas placed in the flask and the solution stirred vigorously. A freshlymade solution of 8N chromic acid was prepared by dissolving chromiumtrioxide (66.7 g, 0.667 mol) in water (40 ml), adding concentratedsulphuric acid (53.3 ml) and adding enough water to bring the solutionvolume to 115 ml. The 8N chromic acid solution (115 ml) was then addeddropwise over a period of 30 min with continued vigorous stirring, thereaction's exotherm being maintained at the optimal temperature of 25°C. by the use of an ice bath. After the complete addition of the chromicacid, the reaction mixture was stirred for a further 15minutes—maintaining the optimal temperature of 25° C. The reactionmixture was then quenched by the addition of methanol (20 ml). Exothermwas controlled by the use of an ice bath and, if necessary, directaddition of a small amount of crushed ice to the reaction mixtureitself. The reaction mixture was filtered through a Celite pad and thenconcentrated in vacuo. The resulting acidic solution was then extractedwith ethyl acetate (3×300 ml) and the combined organic layers washedwith brine (2×100 ml), then dried with magnesium sulfate andconcentrated in vacuo. The crude product was recrystallized from ethylacetate to give a white crystalline product,(2S)-1-(tert-butoxycarbonyl)-4-oxo-2-pyrrolidinecarboxylic acid (22.55g, 76%) (1a). (1H NMR (360 MHz, CDCl3): 1.4 (m, 9H), 2.5–3.0 (m, 2H),3.7–3.9 (m, 2H), 4.75 (dd, 1H)).

Intermediate 1b:(4EZ)-1-(tert-butoxycarbonyl)-4-(methoxyimino)-L-proline

Introduction of the Oxime Moiety:

A solution was made containing(2S)-1-(tert-butoxycarbonyl)-4-oxo-2-pyrrolidine-carboxylic acid(Intermediate 1a, 5.0 g, 21 mmol) and O-methylhydroxylaminehydrochloride (2.7 g, 32.8 mmol) in chloroform (100 ml) containingtriethylamine (5.5 g, 55 mmol). The reaction mixture was then stirred atambient temperature overnight, prior to removal of solvent. Theresultant crude reaction mixture was dissolved in ethyl acetate (150 ml)and washed rapidly with 1N HCl (40 ml). The acidic layer was thenextracted with ethyl acetate (3×20 ml) and the combined organic layerswashed with brine before drying over magnesium sulfate, filtering andremoval of solvent in vacuo. The desired product (1b) (5.3 g, 94%) wasisolated as a pale yellow oil.¹H NMR (400 MHz, CDCl₃); 1.45 (m, 9H),2.8–3.2 (m, 2H), 3.9 (s, 3H), 4.2 (m, 2H), 4.5–4.7 (m, 1H).

Intermediate 1c: 1-tert-butyl-2-methyl(2S,4EZ)-4-(methoxyimino)pyrrolidine-1,2-dicarboxylate.

A solution of the oximether(2S,4EZ)-1-(tert-butoxycarbonyl)-4-(methoxyimino)-2-pyrrolidinecarboxylicacid (intermediate 1b, 0.648 g, 2.5 mmol), in a 1:1 mixture of methanoland toluene (35 ml) was made. Trimethylsilyl diazomethane (3.8 ml of a2M solution in hexanes, 7.5 mmol) was then added dropwise to the stirredsolution at room temperature under nitrogen. After completion of theevolution of nitrogen gas, the resulting yellow solution was evaporatedin vacuo, and the residue filtered through a pad of silica gel, elutingwith ethyl acetate. Removal of solvent from the filtrate gave themethylester (1c) product as a yellow oil (0.646 g, 95% yield).

Intermediate 1d: Methyl(2S,4EZ)-4-(methoxyimino)-1-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]-2-pyrrolidinecarboxylate.

Coupling the R³ Moitey:

A solution was made containing 1-tert-butyl-2-methyl(2S,4EZ)-4-(methoxyimino)-1,2-pyrrolidine-dicarboxylate (intermediate1c, 0.892 g, 3.28 mmol) in anhydrous DCM (28 ml). TFA (20%, 7 mL) wasadded drop wise. The mixture was stirred at r.t. for 20 min. Solventswere evaporated and the desired product, (0.564 g, quant.) was isolatedas a yellow oil. It was directly dissolved in a 7:3 mixture of DCM andDMF (30 ml) and treated with 2′-methyl[1,1′-biphenyl]-4-carboxylic acid(0.765 g, 3.60 mmol) and 4-dimethylaminopyridine (0.880 g, 7.21 mmol).EDC (0.691 mg, 3.60 mmol) was added slowly at 0° C. and the reactionmixture was stirred overnight at rt. It was washed with water (twice 20ml), dried over MgSO₄, filtrated and evaporated in vacuo.

Intermediate 1e: (2S,4EZ)-4-(methoxyimino)-1-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]-2-pyrrolidinecarboxylicacid.

Introduction of the R₂ Moiety:

Methyl(2S,4EZ)-4-(methoxyimino)-1-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]-2-pyrrolidinecarboxylate(intermediate 1d, 391 mg, 1.06 mmol) was stirred at rt for 4 h in asolution containing dioxan (9 ml), water (3 ml) and NaOH (1.13 ml of a1.6 N solution). Dioxan was removed in vacuo and the solution was madeacidic by treatment with HCl 0.1 N. It was extracted with EtOAc, washedwith brine, dried over magnesium sulfate, filtered and concentrated togive the desired product (1e) (342 mg, yield=91%). 1H NMR (300 MHz,CDCl3): 2.23 (s, 1.5H), 2.25 (s, 1.5H), 3.10 (m, 2H), 3.83 (s, 1.5H),3.85 (s, 1.5H), 4.10 (m, 2H), 5.18 (m, 1H), 7.18 (m, 4H), 7.37 (m, 2H),7.57 (m, 2H).

MS (APCI+): 353 (M+1) (APCI−): 351 (M+1).

Example I:(2S,4EZ)-4-(methoxyimino)-1-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]-2-pyrrolidinecarboxylicacid (intermediate 1e, 50 mg, 0.14 mmol) was dissolved in THF (1 ml) andtreated with ethylchloroformate (163 μl, 0.17 mmol) and TEA (29 μl, 0.76mmol) at −15° C. The reaction mixture was stirred at this temperatureand under nitrogen atmosphere for 30 mn before the addition of sodiumborohydride (13.4 mg in 0.65 ml water, 0.35 mmol). It was then allowedto warm to rt. After 3 h, the reaction was quenched with 2.5 ml of a 1 NHCl solution, and extracted with EtOAc three times. The combined organiclayers were washed with a 0.1N HCl solution (three times), water (threetimes), dried over magnesium sulfate, filtered and concentrated to givecompound (1).

Yield: 12% (6 mg)

Appearance: yellow oil

MS (APCI+): 339 (M+1)

HPLC purity: 90.1%

Example II (3EZ,5S-1-(1,1′-biphenyl-4-ylcarbonyl)-5 (hydroxymethyl)pyrrolidin-3-one O-methyloxime (2),(3Z,5S)-1-(1,1′-biphenyl-4-ylcarbonyl)-5 (hydroxymethyl)pyrrolidin-3-oneO-methyloxime (3),(3E,5S)-1-(1,1′-biphenyl-4-ylcarbonyl)-5-(hydroxymethyl)pyrrolidin-3-one O-methyloxime (4) (Compounds of Formula II or Iawherein R² is H)

Intermediate 2a: methyl(4R)-1-(1,1′-biphenyl-4-ylcarbonyl)-4-hydroxy-L-prolinate (Compound ofFormula VIII).

To a solution of 4-biphenylcarboxylic acid (17.5 g, 88.3 mmol) in DMF(100 ml) were added EDC (16.9 g, 88.3 mmol), HOBt (11.9 g, 88.3 mmol)and DIEA (27.9 ml, 183.9 mmol). The mixture was then stirred at r.t. for10 mn before the addition of trans-hydroxy-L-proline methylesterhydrochloride (10.7 g, 73.6 mmol) and left for another 48 h at r.t.under nitrogen atmosphere. It was then concentrated under high vacuumand dissolved in Ethyl acetate, washed with water, 1N chlorhydric acidsolution, saturated sodium hydrogenocarbonate solution and brine. It wasfinally dried over magnesium sulfate, filtered and concentrated. Thecrude thus obtained was purified by flash-chromatography withcyclohexane/ethyl acetate 90:10 (compound 2a).

Yield: 53% (12.6 g)

Appearance: brown solid

1H NMR (CDCl3): 2.11 (m, 1H), 2.36 (m, 1H), 3.58 (d, J=11.5 Hz, 1H),3.77 (s, 3H), 3.86 (dd, J=3.4 and 11.1 Hz, 1H), 4.51 (s, 1H), 4.86 (t,J=8.3 Hz, 1H), 7.33–7.62 (m, 9H).

MS (APCI+): 651 (2M+1).

Intermediate 2b:(3R,5S)-1-(1,1′-biphenyl-4-ylcarbonyl)-5-(hydroxymethyl)-pyrrolidin-3-ol.

Lithium borohydride (600 mg, 25.8 mmmmol) was slowly added to a solutionof methyl ester (2a) (5.6 g, 17.2 mmol) in THF (80 mL) at 0° C. undernitrogen atmosphere. The mixture was stirred at r.t. for 2h and theborohydride neutralized with water. The white precipitate containingcompound (2b) was filtered and washed with ether.

Yield: 82% (4.2 g)

Appearance: white solid

1H NMR (DMSO): 1.90–2.02 (m, 2H), 3.24–3.30 (m, 2H), 3.57 (m, 2H), 3.67(m, 1H), 4.18 (m, 1H), 4.28 (m, 1H), 4.80 (brs, 1H), 7.39–7.74 (m, 9H).APCI (+): 299 (M+1)

Intermediate 2c: (3R,5S)-1-(1,1′-biphenyl-4-ylcarbonyl)-5-({[tert-butyl(dimethyl) silyl]oxy}methyl)pyrrolidin-3-ol (Compound of Formula VII).

A solution of diol (2b) (4.2 g, 14.1 mmol) and TBDMS-Cl (1.9 g, 12.6mmol) in DMF (40 ml) was diluted with DCM (150 ml) and treated with DBU(421 μl, 2.81 mmol) and TEA (1.96 ml, 14.1 mmol). The reaction mixturewas then allowed to stir for 16 h at r.t. under nitrogen atmosphere.After dilution with ethyl acetate, the organic phase was washed withwater. Aqueous phase was extracted again with ethyl acetate and thecombined organic phases were washed with saturated ammonium chloridsolution and three times with brine before being dried over magnesiumsulfate, filtered and concentrated. The crude thus obtained was purifiedby flash chromatography with DCM/MeOH 95:5 (compound 2c).

Yield: 75% (4.39 g)

Appearance: white powder

1H NMR (DMSO): 0.03 (s, 6H), 0.88 (s, 9H), 1.92–2.04 (m, 2H), 3.30 (m,1H), 3.54 (m, 1H), 3.72 (brd, J=9.0 Hz, 1H), 3.92 (m, 1H), 4.20 (m, 1H),4.30 (m, 1H), 4.83 (m, 1H), 7.37–7.56 (m, 5H), 7.77 (m, 4H).

LC/MS (ESI, +): 412 (M+1).

Intermediate 2d: (5S)-1-(1,1′-biphenyl-4-ylcarbonyl)-5-({[tert-butyl(dimethyl) silyl]oxy}methyl)pyrrolidin-3-one (Compound of Formula IV).

A solution of dry DMSO (2.04 ml, 28.8 mmol) in DCM (15 ml) was slowlyadded to a solution of oxalyl chlorid (1.34 ml, 15.7 mmol) in DCM (5 mL)at −78° C. under nitrogen atmosphere. The mixture was allowed to stirfor 30 mn before the slow addition of alcohol (2c) (5.38 g, 13.1 mmmol)in DCM (50 ml). The reaction mixture was stirred for 3 h at −78° C.,treated dropwise with TEA (9.06 ml, 65.3 mmol) and allow to warm to r.t.It was then washed with brine, 1N HCl solution, with brine again, driedover magnesium sulfate, filtered and concentrated Compound 2d.

Yield: 91% (4.88 g)

Appearance: brown oil

1H NMR (CDCl3): 0.06 (s, 6H), 0.86 (s, 9H), 2.49–2.7θ (m, 2H), 3.69 (m,1H), 3.84 (m, 1H), 3.98 (m, 1H), 4.20 (m, 1H), 5.07 (m, 1H), 7.37–7.65(m, 9H).

LC/MS (ESI,+): 410 (M+1).

Intermediate 2e: (3EZ,5S)-1-(1,1′-biphenyl-4-ylcarbonyl)-5-({[tert-butyl(dimethyl) silyl]oxy}methyl)pyrrolidin-3-one O-methyloxime.

A mixture of ketone 2d (4.78 g, 11.7 mmol), methylhydroxylaminehydrochlorid (2.44 g, 29.2 mmol) and TEA (4.05 ml, 29.2 mmol) inchloroform (80 ml) is heated at 65° C. for 16 h. The mixture is thenwashed with brine, 1N HCl solution, brine again and dried over magnesiumsulfate, filtered and concentrated to give compound 2e.

Yield: 86% (4.41 g)

Appearance: brown oil

1H NMR (CDCl3): 0.06 (s, 6H), 0.88 (s, 9H), 2.68–2.90 (m, 2H), 3.42 (m,1H), 3.78 (s, 1.5H), 3.83 (s, 1.5H), 4.1 (m, 2H), 4.31 (m, 1H), 4.83 (m,1H), 7.34–7.64 (m, 9H).

LC/MS: ESI (+): 439 (M+1)

Final Compounds: (3EZ,5S)-1-(1,1′-biphenyl-4-ylcarbonyl)-5(hydroxymethyl) pyrrolidin-3-one O-methyloxime (2),(3Z,5S)-1-(1,1′-biphenyl-4-ylcarbonyl)-5 (hydroxymethyl)pyrrolidin-3-oneO-methyloxime (3),(3E,5S)-1-(1,1′-biphenyl-4-ylcarbonyl)-5-(hydroxymethyl)pyrrolidin-3-oneO-methyloxime (4) (compounds of formula II or of formula Ia when R² isH):

A solution of TBAF (14.1 ml of a solution 1M in THF, 14.1 mmol) wasadded to a solution of oxime (2e) (4.13 g, 9.41 mmol) in THF (100 ml).The reaction mixture was allowed to stir at room temperature overnight.It was then concentrated and diluted with ethyl acetate. Organic phasewas washed with water, 1H HCl solution and brine before being dried overmagnesium sulfate, filtered and concentrated.

Yield: quantitative of the EZ mixture (2)

Appearance: white foam

LC/MS: ESI (+): 325 (M+1)

The two isomers E and Z were separated by flash chromatography usingethyl acetate/cyclohexane 80:20 as eluant.

Less polar fraction:(3E,5S)-1-(1,1′-biphenyl-4-ylcarbonyl)-5-(hydroxymethyl)pyrrolidin-3-one O-methyloxime (3)

Rf: 0.36 (AcOEt/cyclohexane 80:20)

Yield: 25% (765 mg)

Appearance: white foam

1H NMR (DMSO): 2.64 (brs, 2H), 3.20–3.7 (m, 3H), 3.80 (s, 3H), 3.8–4.6(m, 2H), 5.00 (t, J=8.0 Hz, 1H), 7.37–7.60 (m, 9H).

IR (film): 3292, 1604,1417, 1040

MS(APCI,+): 325 (M+1)

Elemental analysis: (C₁₉H₂₀N₂O₃;0.2H₂O): calc.: C: 69.58; H: 6.27; N:8.54; exp.: C: 69.53; H: 6.32; N: 8.36

HPLC purity: 98.6%

More polar fraction:(3Z,5S)-1-(1,1′-biphenyl-4-ylcarbonyl)-5-(hydroxymethyl)pyrrolidin-3-one O-methyloxime (4)

Rf: 0.22 (AcOEt/cyclohexane 80:20)

Yield: 33% (1013 mg)

Appearance: white powder

Melting point: 189° C.

1H NMR (DMSO): 2.64–2.82 (m, 2H), 3.20–3.57 (m, 3H), 3.70–3.80 (m, 3H),3.98–4.60 (m, 2H), 5.0 (t, J=8.0 Hz, 1H), 7.37–7.76 (m, 9H).

IR (film): 3373,1606,1417,1045

MS (APCI,+): 649 (2M+1), 325 (M+1)

Elemental analysis: (C₁₉H₂₀N₂O₃): calc.: C: 70.35; H: 6.21; N: 8.64;exp.: C: 70.22; H: 6.27; N: 8.56

HPLC purity: 99.9%

Note: a fraction of E/Z mixture (470 mg) was isolated as well.

Example III tert-butyl{[(2S,4EZ)-1-(1,1′-biphenyl-4-ylcarbonyl)-4-(methoxyimino)pyrrolidin-2-yl]methoxy}acetate (5) (Compound of Formula Ia)

To a stirred solution of alcohol (2) (EZ mixture, 58 mg, 0.18 mmol) andtert-butyl bromoacetate (530 μl, 3.6 mmol) in dichloromethane (0.2 ml)were added 50% aqueous NaOH (0.8 ml) and tetrabutylammonium chloride (50mg, 0.18 mmol) at room temperature, and the whole reaction mixture wasstirred for 1 hour. After dilution with water, the mixture was extractedwith ethyl acetate, organic phase was washed with brine, dried (MgSO₄)and concentrated. The product (compound 5) was purified by silica gelcolumn chromatography using DCM:MeOH, 95:5 as eluant.

Yield: 99% (85 mg)

LC/MS (ESI, −): 381 (M-tBu-H)

¹H NMR (CDCl₃): 1.45 (s, 9H), 2.94 (m, 2H), 3.60–4.20 (m, 8H), 4.36 (m,1H), 4.90 (m, 1H), 7.30–7.70 (m, 9H).

HPLC purity: 92%

Example IV{[(2S,4EZ)-1-(1,1′-biphenyl-4ylcarbopyl)-4-(methoxyimino)pyrrolidin-2-yl]methoxy}aceticacid (6) (Compound of Formula Ia)

To a solution of tert-butyl ester (5) (45 mg, 0.1 mmol) indichloromethane (0.5 ml) was added trifluoroacetic acid (0.1 ml) at roomtemperature. Once the reaction was completed, the mixture wasconcentrated in vacuo. The crude was dissolved in dichloromethane andwashed with HCl 1M. The organic layer was dried (MgSO₄) andconcentrated.

Yield: 40% (20 mg)

LC/MS (ESI−): 381 (M−H) (ESI+): 383 (M+H)

HPLC purity: 74%

Example V2-{[(2S,4EZ)-1-(1,1′-biphenyl-4-ylcarbonyl)-4-(methoxyimino)pyrrolidin-2-yl]methoxy}-N-(2-pyrrolidin-1-ylethyl)acetamide(7) (Compound of Formula Ia)

A solution of acid (6) (15 mg, 0.04 mmol), 1-(2-aminoethyl)-pyrrolidine(6 μl, 0.05 mmol), DIC (7.2 μl, 0.05 mmol) and DMAP (1 mg, 0.01 mmol) indichloromethane (1 ml) was stirred under argon at room temperature for18 hours. The mixture was concentrated in vacuo and purified on silicagel preparative chromatography using DCM: MeOH, 50:50 as eluant.

Yield: 80% (17 mg)

¹H NMR (CDCl₃): 1.71 (s, 4H), 2.30–4.00 (m, 8H), 3.37 (m, 2H), 3.50–4.40(m, 9H), 4.96 (m, 1H), 6.87 (m, 1H), 7.30–7.70 (m, 9H)

LC/MS (ESI−): 477 (M−H) (EST+): 479 (M+H)

HPLC purity: 88%

Example VI(3EZ,5S)-1-(1,1′-biphenyl-4-ylcarbonyl)-5-(methoxymethyl)pyrrolidin-3-oneO-methyloxime (8) (Compound of Formula Ia)

To a solution of alcohol (2) (EZ mixture, 20 mg, 0.06 mmol) and sodiumhydride (3 mg, 0.12 mmol) in tetrahydrofuran (1 ml) under argon, wasadded methyl iodide (7.7 μl, 0.12 mmol). The reaction mixture wasstirred overnight and quenched with water. The mixture was diluted withethyl acetate and washed with brine, dried (MgSO₄) and concentrated invacuo. The crude was purified on silica gel preparative chromatographyusing DCM: MeOH 100:0 then 95:5.

Yield: 94% (21 mg)

¹H NMR (CDCl₃): 2.80 (m, 2H), 3.35 (m, 3H), 3.46 (m, 1H), 3.67 (m, 1H),3.84 (s, 3H), 4.27 (m, 2H), 4.91 (m, 1H), 7.30–7.70 (m, 9H).

LC/MS (ESI+): 339 (M+1)

HPLC purity: 93%

Example VII(3EZ,5S)-1-(1,1′-biphenyl-4-ylcarbonyl)-5-[(4-methylpiperazin-1-yl)methyl]pyrrolidin-3-oneO-methyloxime (9) (Compound of Formula Ib)

Intermediate 9a: [(2S,4EZ)-1-(1,1′-biphenyl-4-ylcarbonyl)-4-(methoxyimino)pyrrolidin-2-yl]methylmethanesulfonate (Compound of Formula IIa).

Mesyl chlorid (48 μl, 0.62 mmol) was added to a solution of alcohol (2)(EZ mixture, 80 mg, 0.25 mmol) in DCM (8 ml) cooled at 0° C. andmaintained under nitrogen atmosphere. The reaction mixture was thenallowed to warm to r.t. and monitored by tlc. Completion was achievedafter 1 h30. Organic phase was washed with saturated ammonium chloridsolution and brine, dried over magnesium sulfate, filtered andconcentrated.

Yield: quant. (115 mg)

HPLC purity: 87%

Example VII: the mesylate (9a) (60 mg, 0.15 mmol) was dissolved inMEK/ACN (1:1, 10 ml) and treated with lithium bromid (16 mg, 0.18 mmol).The reaction mixture was heated at 85° C. before the addition ofN-methylpiperazine (22 mg, 0.22 mmol) and TEA (31 μl, 0.22 mmol) andstirred at this temperature overnight. It was then concentrated,redissolved in Ethyl Acetate and washed with saturated NaHCO₃ solution,brine, dried over magnesium sulfate, filtered and concentrated. Thecrude (48 mg) was finally purified by flash chromatography withDCM/MeOH/NH₄OH 92:8:1 to give compound 9a.

Rf: 0.17 (DCM/MeOH/NH₄OH 90:10:1)

Yield: 36% (22 mg)

Appearance: brown oil

1H NMR (CDCl3): 2.31 (s, 3H), 2.45–2.86 (m, 14H), 3.85 (brs, 3H), 4.13(m, 1H), 7.34–7.64 (m, 9H).

LC/MS (ESI, +): 407 (M+1)

HPLC purity: 97.1%

Example VIII(3EZ,5S)-1-(1,1′-biphenyl-4-ylcarbonyl)-5-{[(4-methoxyphenyl)amino]methyl}pyrrolidin-3-one O-methyloxime (10) (Compound of FormulaIb)

A solution of mesylate (9a) (32 mg, 0.08 mmol), p-methoxy aniline (20mg, 0.16 mmol) and triethylamine (22 μl, 0.16 mmol) in methyl ethylketone/acetonitrile (2 ml, 1:1) was stirred for 2 days. The reactionmixture was diluted with ethyl acetate and washed wit NH₄Cl sat. Theorganic phase was dried (MgSO₄) and concentrated. The crude thusobtained was purified by HPLC using a PARALLEX FLEX® system.

Yield: 21% (10 mg)

HPLC purity: 72%

LC/MS (ESI+): 430 (M+1)

Example IX(3EZ,5S)-1-(1,1′-biphenyl-4-ylcarbonyl)-5-({[2-(1H-pyrazol-1-yl)ethyl]amino}methyl)pyrrolidin-3-one O-methyloxime (11) (Compound ofFormula Ib)

A solution of mesylate (9a) (60 mg, 0.15 mmol), 1-(2′-aminoethyl)pyrazole (58 mg, 0.53 mmol), potassium carbonate (41 mg, 0.30 mmol) andsodium iodide (225 mg, 1.50 mmol) in tetrahydrofuran (5 ml) was stirredfor 2 days. The reaction mixture was diluted with ethyl acetate andwashed with HCl 1N, then with brine. The organic phase was dried (MgSO₄)and concentrated. The crude was purified using a C8 SPE cartridge.

Yield: 5% (3 mg).

LC/MS (ESI+): 418 (M+H)

HPLC purity: 79%.

Example X2-{[(2S,4EZ)-1-(1,1′-biphenyl-4-ylcarbonyl)-4-(methoxyimino)pyrrolidin-2-yl]methyl}-1H-isoindole-1,3(2H)-dione(12) (Compound of Formula Ib)

A solution of alcohol (2) (EZ mixture, 51 mg, 0.16 mmol), phtalimide (70mg, 0.48 mmol), triphenylphosphine polymer bound (158 mg, 0.48 mmol) anddiethyl azodicarboxylate (40% in toluene, 205 μl, 0.48 mmol) intetrahydrofuran (5 ml) was stirred for 2 days. The resin was filteredoff and the reaction mixture was concentrated in vacuo. The crude waspurified on silica gel preparative chromatography using DCM as eluant.

Yield: 59% (50 mg).

¹H NMR (CDCl₃): 2.76 (m, 2H), 3.60–4.50 (m, 7H), 5.32 (m, 1H), 7.20–8.00(m, 13H).

LC/MS (ESI+): 454 (M+1)

HPLC purity: 85%

Example XI(3EZ,5S)-5-(aminomethyl)-1-(1,1′-biphenyl-4-ylcarbonyl)pyrrolidin-3-oneO-methyloxime (13) (Compound of Formula III)

A solution of phtalimide (12) (42 mg, 0.09 mmol), hydrazine monohydrate(45 μl, 0.93 mmol) in ethanol: tetrahydrofuran (1:1, 1 ml) was stirredovernight. The white precipitate was filtered off and the filtrate wasconcentrated in vacuo to give the expected amine.

Yield: 76% (26 mg)

LC/MS (ESI−): 422 (M−1) (ESI+): 424 (M+1)

¹H NMR (CDCl₃): 2.29 (m, 1H), 2.70 (m, 1H), 3.43 (m, 1H), 3.64 (m, 3H),3.83 (s, 3H), 4.17 (m, 1H), 6.90 (m, NH2), 7.20–8.00 (m, 9H).

HPLC purity: 88%.

Example XIIN-{[(4EZ,2S)-1-(1,1′-biphenyl-4-ylcarbonyl)-4-(methoxyimino)pyrrolidin-2-yl]methyl}acetamide(14) (Compound of Formula Ib)

A solution of amine (13) (16 mg, 0.05 mmol), acetic anhydride (5.6 μl,0.06 mmol) and triethylamine (7.9 μl, 0.06 mmol) in dichloromethane wasstirred 30 min. The reaction mixture was washed with water. The organicphase was dried (MgSO₄) and concentrated in vacuo. The crude waspurified on silica gel preparative chromatography using Ethyl acetate aseluant.

Yield: 42% (8 mg).

LC/MS (ESI−): 364 (M−1) (ESI+): 366 (M+1)

¹H NMR (CDCl₃): 2.10 (s, 3H), 2.40–3.00 (m, 2H), 3.54 (m, 2H), 3.87 (s,3H), 4.24 (m, 2H), 4.81 (m, 1H), 7.20–8.00 (m, 9H).

HPLC purity: 95%.

Example XIII(3EZ,5S)-1-(1,1′-biphenyl-4-ylcarbonyl)-5-(piperidin-1-ylmethyl)pyrrolidin-3-one O-methyloxime (15) (Compound of Formula Ib)

Intermediate 15a: methyl(4R)-1-(1,1′-biphenyl-4-ylcarbonyl)-4-{[tert-butyl (dimethyl)silyl]oxy}-L-prolinate (Compound of Formula VIII).

Methyl (4R)-1-(1,1′-biphenyl-4-ylcarbonyl)-4-hydroxy-L-prolinate(intermediate 2a, 2.07 g, 6.35 mmol) was dissolved in DCM (30 ml) andtreated with 4-DMAP (776 mg, 6.35 mmol), TEA (2.21 ml, 15.88 mmol) andTBDMS-Cl (1.91 g, 12.7 mmol). The reaction was monitored by LC/MS. After24 h, as the reaction was not completed, TBDMS-CL (300 mg, 2 mmol) andTEA (1 ml) were added. After 48 h, the reaction was completed. Themixture was washed with sat. NH₄Cl and brine (twice), dried overmagnesium sulfate, filtered and concentrated. The crude (2.85 g) waspurified by flash chromatography using EtOAc/cHex 50:50 as eluant.

Yield: 93% (2.61 g)

1H NMR (CDCl3): −0.05 (s, 3H), 0.02 (s, 3H), 0.81 (s, 9H), 2.04 (m, 1H),2.27 (m, 1H), 3.45 (d, J=9.4 Hz, 1H), 3.78 (s, 3H), 3.81 (m, 1H), 4.43(m, 1H), 4.80 (t, J=8.1 Hz, 1H), 7.33–7.46 (m, 3H), 7.62 (m, 6H).

Intermediate 15b: ((2S,4R)-1-(1,1′-biphenyl-4-ylcarbonyl)-4-{[tert-butyl(dimethyl) silyl]oxy}pyrrolidin-2-yl)methanol.

A solution of methyl (4R)-1-(1,1′-biphenyl-4-ylcarbonyl)-4-{[tert-butyl(dimethyl) silyl]oxy}-L-prolinate (intermediate 15a, 2.61 g, 5.94 mmol)in THF (60 ml) was cooled to 0° C. and treated with lithium borohydride(95%, 206 mg, 8.9 mmol). The reaction mixture was stirred for 3 h andquenched slowly with water. THF was removed under reduced pressure, thecrude was redissolved in AcOEt, washed with sat. NH₄Cl, brine, driedover magnesium sulfate, filtered and concentrated.

Yield: 92% (2.268 g)

LC/MS (ESI+): 412 (M+1)

Intermediate 15c:1-[((2S,4R)-1-(1,1′-biphenyl-4-ylcarbonyl)-4-{[tert-butyl (dimethyl)silyl]oxy}pyrrolidin-2-yl)methyl]piperidine (Compound of Formula XI)

To a solution of alcohol (15b) (200 mg, 0.49 mmol) in dichloromethane (5ml) under argon, was added Dess Martin reagent (227 mg, 053 mmol). Thereaction mixture was stirred for 24 hours, then was diluted withdichloromethane and washed with NaHCO₃ sat. The aqueous layer wasextracted with dichloromethane. The organic phases were washed withwater, dried (MgSO₄) and concentrated. The aldehyde obtained wasdirectly engaged in the following step. To a solution of aldehyde (184mg, 0.45 mmol) in 1,2-dichloroethane were added piperidine (49 μl, 0.50mmol), acetic acid (28 μl, 0.50 mmol) and then sodiumtriacetoxyborohydride (143 mg, 0.68 mmol). The reaction was stirred overnight and then diluted with ethyl acetate. The organic phase was washedwith sat. NaHCO₃, then with brine. The organic phase was dried (MgSO₄)and concentrated to afford the expected tertiary amine.

Yield: 95% (230 mg).

LC/MS (ESI−): 513 (M+Cl) (ESI+): 479 (M+1)

¹H NMR (CDCl₃): −0.09 (s, 3H), 0.00 (s, 3H), 0.79 (s, 9H), 1.20–1.60 (m,6H), 2.08 (m, 4H), 2.40–2.90 (m, 4H), 3.35 (m, 1H), 3.56 (m, 1H), 4.34(m, 1H), 4.59 (m, 1H), 7.20–7.60 (m, 9H).

HPLC purity: 90%.

Intermediate 15d:(3R,5S)-1-(1,1′-biphenyl-4-ylcarbonyl)-5-(piperidin-1-ylmethyl)pyrrolidin-3-ol (Compound of Formula XI)

A solution of protected alcohol (15c) (200 mg, 0.42 mmol) and TBAF (0.63ml, 1M in THF) in tetrahydrofuran was stirred at room temperature during1 hour. The reaction mixture was concentrated and then diluted inacetone-ethyl acetate (1-2) and washed with a saturated NaHCO₃ solution.The organic phase was dried (MgSO₄) and concentrated to afford theexpected alcohol.

Yield: 51% (90 mg).

LC/MS (ESI+): 365 (M+1)

¹H NMR (CDCl₃): 1.20–1.60 (m, 6H), 2.19 (m, 4H), 2.25–2.90 (m, 4H), 3.50(m, 2H), 4.43 (m, 1H), 4.62 (m, 1H), 7.30–7.70 (m, 9H).

HPLC purity: 86%.

Intermediate 15e:(5S)-1-(1,1′-biphenyl-4-ylcarbonyl)-5-(piperidin-1-ylmethyl)-pyrrolidin-3-one(Compound of Formula X)

A solution of DMSO (46.8 μl, 0.66 mmol) in dichloromethane (1 ml) wasadded drop wise to a solution of oxalyl chloride (28.2 μl, 0.33 mmol) indichloromethane (2 ml) at −78° C. under argon. After 15 min at −78° C.,a solution of alcohol (15d) (80 mg, 0.22 mmol) in dichloromethane (1 ml)was added dropwise. The reaction mixture was stirred at −78° C. for 1hour, treated with triethylamine (0.152 ml, 1.1 mmol) and allowed towarm to room temperature. The reaction mixture was diluted with ethylacetate, washed with water then brine. The organic phase was dried(MgSO₄) and concentrated to afford the expected ketone.

Yield: 84% (78 mg).

LC/MS (ESI−): 361 (M−1) (ESI+): 363 (M+1)

HPLC purity: 86%.

Example XIII: A solution of ketone (15e) (70 mg, 0.19 mmol),hydroxylamine methyl ether hydrochloride (48 mg, 0.58 mmol) andtriethylamine (80 μl, 0.58 mmol) in chloroforme (3 ml) was stirred at70° C. for 2 days. The reaction mixture was diluted with dichloromethaneand washed with HCl 1N. The organic phase was dried (MgSO₄) andconcentrated to afford the oxime ether.

Yield: 90% (73 mg).

LC/MS (ESI+): 392 (M+1)

¹H NMR (CDCl₃): 1.39 (m, 2H), 1.50–1.90 (m, 4H), 2.07 (m, 2H), 2.43 (m,2H), 2.50–3.10 (m, 4H), 3.38 (m, 1H), 3.68 (m, 1H), 3.79 (m, 3H), 4.21(m, 2H), 4.86 (m, 1H), 7.20–8.00 (m, 9H).

HPLC purity: 94%.

Example XIV(3EZ,5S)-1-(1,1′-biphenyl-4-ylcarbonyl)-5-(2-hydroxyethyl)pyrrolidin-3-oneO-methyloxime (16) (Compound of Formula II)

Intermediate 16a:[(2S,4R)-1-(1,1′-biphenyl-4-ylcarbonyl)-4-hydroxypyrrolidin-2-yl]aceticacid (Compound of Formula VIII)

To a solution of commercial L-beta-homohydroxyproline hydrochloride (245mg, 1.35 mmol), triethylamine (1.13 ml, 8.09 mmol) in water (0.8 ml) andtetrahydrofuran (2 ml) at 0° C. under argon, was added dropwise asolution of 4-phenylbenzoyl chloride (438 mg, 2.02 mmol) intetrahydrofuran (1 ml). The reaction mixture was allowed to warm to roomtemperature and was stirred 18 hours. It was then diluted withacetone-ethyl acetate (1-2) and washed with HCl 1N. The organic phasewas dried (MgSO₄) and concentrated to afford a mixture of desiredproduct and 4-phenylbenzoyl acid. A small quantity of acid could beobtained by precipitation with ethyl acetate.

¹H NMR (DMSO): 1.82 (m, 1H), 2.11 (m, 1H), 2.5 (m, 1H), 2.81 (dd, J=15.6Hz, J=3.2, 1H), 3.3 (m, 1H), 3.51 (dd, J=11.7 Hz, J=2.6 Hz, 1H), 4.16(m, 1H), 4.40 (m, 1H), 7.30–8.00 (m, 9H).

LC/MS: (ESI−): 280 (M−1CO₂), 324 (M−1) (ESI+): 326 (M+1)

HPLC purity: 84%

Intermediate 16b: methyl[(2S,4R)-1-(1,1′-biphenyl-4-ylcarbonyl)-4-hydroxy-pyrrolidin-2-yl]acetate(Compound of Formula VIII)

To a solution of the acid mixture previously obtained (intermediate 16a)in toluene-methanol (10 ml, 1-1) was added diazomethyltrimethylsilane(2.76 ml, 2M in hexane). After 3 hours, the reaction mixture wasconcentrated and purified by silica gel column chromatography usingethyl acetate as eluant.

Yield: 40% (for the two steps, 256 mg).

¹H NMR (DMSO): 1.82 (m, 1H), 2.11 (m, 1H), 2.6 (dd, J=15.4 Hz, J=8.3 Hz,1H), 2.97 (dd, J=15.3 Hz, J=3.4 Hz, 1H), 3.25 (d, J=11.4 Hz, 1H), 3.62(s, 3H), 3.67 (dd, J=11.4 Hz, J=3.4 Hz, 1H), 4.16 (m, 1H), 4.44 (m, 1H),4.86 (d, J=3.4 Hz, OH) 7.30–8.00 (m, 9H).

LC/MS (ESI+): 340 (M+1)

HPLC purity: 98%.

Intermediate 16c:(3R,5R)-1-(1,1′-biphenyl-4-ylcarbonyl)-5-(2-hydroxyethyl)-pyrrolidin-3-ol(Compound of Formula VII)

To a solution of methyl ester (16b) (310 mg, 0.91 mmol) intetrahydrofuran at 0° C. umder argon, was added lithium borohydride (30mg, 1.37 mmol). The reaction mixture was allowed to warm at roomtemperature and stirred 12 h. LiBH₄ was quenched with water and thetetrahydrofuran evaporated in vacuo. Acetonitrile was added and thewhite precipitate was filtered, washed with acetonitrile then with etherand finally dried.

Yield: 97% (280 mg)

¹H NMR (DMSO): 1.69 (m, 1H), 1.87 (m, 1H), 2.15 (m, 2H), 3.35 (m, 1H),3.57 (m, 2H), 3.72 (d, J=11.3 Hz, 1H), 4.25 (m, 1H), 4.40 (m, 1H), 4.56(m, OH), 4.87 (m, OH) 7.30–8.00 (m, 9H).

LC/MS (ESI+): 294 (M−H₂O+1), 312 (M+1)⁺, 334 (M+Na)

HPLC purity: 98.5%.

Intermediate 16d:(3R,5R)-1-(1,1′-biphenyl-4-ylcarbonyl)-5-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)pyrrolidin-3-ol (Compound of Formula VII).

To a solution of diol (16c) (270 mg, 0.87 mmol) in dimethylformamide (10ml) was added dropwise a solution of tert-butyldimethylsilyl chloride(131 mg. 0.87 mmol) and triethylamine (120 μl, 0.87 mmol). The reactionmixture was stirred at room temperature for 2 days. Ethyl acetate wasadded and the reaction mixture was washed with water. The aqueous phasewas extracted with ethyl acetate. The organic phases were dried (MgSO₄)and concentrated in vacuo. The crude was purified on silica gelpreparative chromatography using Ethyl acetate: cyclohexane, 50:50 aseluant. Yield: 17% (63 mg).

LC/MS (ESI+): 426 (M+1)

HPLC purity: 100%.

Intermediate 16e: (5R)-1-(1,1′-biphenyl-4-ylcarbonyl)-5-(2-{[tert-butyl(dimethyl) silyl]oxy}ethyl)pyrrolidin-3-one (Compound of Formula IV).

A solution of DMSO (31.4 μl, 0.44 mmol) in dichloromethane (1 ml) wasadded drop wise to a solution of oxalyl chloride (19 μl, 0.22 mmol) indichloromethane (2 ml) at −78° C. under argon. After 15 min at −78° C.,A solution of alcohol (16d) (63 mg, 0.15 mmol) in dichloromethane (1 ml)was added dropwise. The reaction mixture was stirred at −78° C. for 1hour and triethylamine (0.102 ml, 0.74 mmol) was added and allowed towarm to room temperature. The reaction mixture was diluted with ethylacetate, washed with water then brine. The organic phase was dried(MgSO₄) and concentrated to afford the expected ketone.

Yield: 100% (64 mg).

LC/MS (ESI−): 422 (M−1) (ESI+): 424 (M+1)

HPLC purity: 99%.

Intermediate 16f:(3Z,5S)-1-(1,1′-biphenyl-4-ylcarbonyl)-5-(2-{[tert-butyl (dimethyl)silyl]oxy}ethyl)pyrrolidin-3-one O-methyloxime.

A solution of ketone (16e) (64 mg, 0.15 mmol), hydroxylamine methylether hydrochloride (38 mg, 0.45 mmol) and triethylamine (62 μl, 0.45mmol) in chloroform (4 ml) was stirred at 70° C. for 5 days. Thereaction mixture was diluted with dichloromethane and washed with HCl1N. The organic phase was dried (MgSO₄) and concentrated to afford theexpected oxime ether.

Yield: 96% (68 mg).

¹H NMR (CDCl₃): 0.01 (s, 6H), 0.84 (s, 9H), 1.5–2.0 (m, 2H), 2.66 (m,1H), 2.81 (m, 1H), 3.67 (m, 2H), 3.82 (s, 3 H), 4.20 (m, 2H), 4.88 (m,1H), 7.20–8.00 (m, 9H).

HPLC purity: 95%.

LC/MS (ESI+): 453 (M+1)

Example XIV: A solution of protected alcohol (16f) (68 mg, 0.15 mmol)and TBAF (0.225 ml, 1M in THF) in tetrahydrofuran was stirred at roomtemperature during 1 hour. The reaction mixture was concentrated andthen diluted in ethyl acetate and washed with water. The organic phasewas dried (MgSO₄) and concentrated.

Yield: 85%.

¹H NMR (CDCl₃): 1.60 (m, 1H), 1.89 (m, 1H), 2.55 (m, 1H), 2.92 (m, 1H),3.67 (m, 2H), 3.82 (s, 3 H), 4.20 (m, 2H), 5.07 (m, 1H), 7.20–8.00 (m,9H).

LC/MS (ESI+): 339 (M+1)

HPLC purity: 96%.

Example XV Preparation of a Pharmaceutical Formulation

The following Formulation examples illustrate representativepharmaceutical compositions according to the present invention being.

Formulation 1—Tablets

A pyrrolidine compound of Formula I is admixed as a dry powder with adry gelatin binder in an approximate 1:2 weight ration. A minor amountof magnesium stearate is added as a lubricant. The mixture is formedinto 240–270 mg tablets (80–90 mg of active pyrrolidine compound pertablet) in a tablet press.

Formulation 2—Capsules

A pyrrolidine compound of Formula I is admixed as a dry powder with astarch diluent in an approximate 1:1 weight ratio. The mixture is filledinto 250 mg capsules (125 mg of active pyrrolidine compound percapsule).

Formulation 3—Liquid

A pyrrolidine compound of Formula I (1250 mg), sucrose (1.75 g) andxanthan gum (4 mg) are blended, passed through a No. 10 mesh U.S. sieve,and then mixed with a previously prepared solution of microcrystallinecellulose and sodium carboxymethyl cellulose (11:89, 50 mg) in water.Sodium benzoate (10 mg), flavor, and color are diluted with water andadded with stirring. Sufficient water is then added to produce a totalvolume of 5 mL.

Formulation 4—Tablets

A pyrrolidine compound of Formula I is admixed as a dry powder with adry gelatin binder in an approximate 1:2 weight ratio. A minor amount ofmagnesium stearate is added as a lubricant. The mixture is formed into450–900 mg tablets (150–300 mg of active pyrrolidine compound) in atablet press.

Formulation 5—Injection

A pyrrolidine compound of Formula I is dissolved in a buffered sterilesaline injectable aqueous medium to a concentration of approximately 5mg/ml.

Example XVI Biological Assays

The compounds according to Formula I may be subjected to the followingassays:

-   -   a) In vitro Competition Binding Assay on hOT Receptor with        Scintillation Proximity Assay (11).

This assay allows to determine the affinity of the test compounds forthe human Oxytocin (hOT) receptor. Membranes from HEK293EBNA (cellsexpressing the hOT receptor) were suspended in buffer containing 50 mMTris-HCl, pH 7.4, 5 mM MgCl2 and 0.1% BSA (w/v). The membranes (2–4 μg)were mixed with 0.1 mg SPA bead coated with wheat-germ aglutinin(WGA-PVT-Polyethylene Imine beads from Amersham) and 0.2 nM of theradiolabelled [¹²⁵I]-OVTA (OVTA being Ornithin Vasoactive, an analogueof OT for competitive binding experiments). Non-specific binding wasdetermined in the presence of 1 μM Oxytocin. The total assay volume was100 μl. The plates (Corning® NBS plate) were incubated at roomtemperature for 30 min and counted on a Mibrobeta® plate scintillationcounter. Competitive binding was performed in presence of compounds offormula (I) at the following concentrations: 30 μM, 10 μM, 1 μM, 300 nM,100 nM, 10 nM, 1 nM, 100 pM, 10 pM. The competitive binding data wereanalysed using the iterative, nonlinear, curve-fitting program, “Prism”(GraphPad Software, Inc).

The ability of pyrrolidine derivatives of formula (I) to inhibit thebinding of ¹²⁵-OVTA to the OT-receptor was assessed using the abovedescribed in vitro biological assay. Representative values for someexample compounds are given in Table I where the binding affinity oftest compounds from the above examples is expressed by the inhibitionconstant (Ki; nM). From these values, it can be derived that said testcompounds according to formula I do show a significant binding to theoxytocin receptor.

TABLE I Binding Com- Affinity pound hOT-R No. IUPAC-Name (Ki [nM]) 2(3EZ,5S)-1-(1,1′-biphenyl-4-ylcarbonyl)-5- 139(hydroxymethyl)pyrrolidin-3-one O- methyloxime 4(3Z,5S)-1-(1,1′-biphenyl-4-ylcarbonyl)-5- 94.9(hydroxymethyl)pyrrolidin-3-one O- methyloxime 72-{[(2S,4Z)-1-(1,1′-biphenyl-4-ylcarbonyl)- 1404-(methoxyimino)pyrrolidin-2-yl]methoxy}-N-(2-pyrrolidin-1-ylethyl)acetamide 8(3EZ,5S)-1-(1,1′-biphenyl-4-ylcarbonyl)-5- 55.0(methoxymethyl)pyrrolidin-3-one O-methyloxime 122-{[(2S,4Z)-1-(1,1′-biphenyl-4-ylcarbonyl)- 5.14-(methoxyimino)pyrrolidin-2-yl]methyl}-1H- isoindole-1,3(2H)-dione 16(3EZ,5S)-1-(1,1′-biphenyl-4-ylcarbonyl)-5- 120(2-hydroxyethyl)pyrrolidin-3-one O-methyloxime

-   -   b) Functional Assay No. 1: Inhibition of Oxytocin Mediated        Ca²⁺-mobilization by FLIPR® (Fluorimetric Imaging Plate Reader)

The action of OT-receptor triggers a complex cascade of events in thecell which leads to an increase in the intra-cytoplasmic Ca²⁺concentration. This increase in Ca²⁺ concentration results from bothcalcium release from the sarcoplasmic reticulum (calcium stores) intothe cytoplasm and from calcium influx from the extracellular spacethrough Ca²⁺ channels. This Ca²⁺ mobilization into the cytoplasmtriggers the contractile machinery of the myometrial cells which leadsto uterine contractions (1 and 3).

This assay allows the measurement of the inhibition of OT/OT-R mediatedcalcium mobilization by test compounds of formula (I).

FLIPR® is a fluorimetric imaging device using a laser (Argon-ion laser)for simultaneous illumination and reading (cooled CCD camera) of eachwell of a 96-well-plate, thus enabling rapid measurements on a largenumber of samples.

Preparing the plates: FLIPR-plates were pre-coated with PLL(Poly-L-Lysine) 10 μg/ml+0.1% gelatine to attach HEK293EBNA cells (HumanEmbryonic Kidney cells expressing the hOT receptor) and incubated for 30min up to 2 days at 37° C. The cells were plated out into 96-well-plates(60000 cells/well).

Labelling with fluo-4: 50 μg of fluo-4 (Ca2+ sensitive fluorescent dye)were dissolved in 20 μl pluronic acid (20% in DMSO). The dissolvedfluo-4 was then diluted in 10 ml DMEM (Dubecco's Minimal EssentialMedium)-F12 culture medium. The plates were washed one time withDMEM-F12 medium. 100 μl of the fluo-4 containing-DMEM-F12 medium wereadded to the HEK-cells which were incubated for 1.5–2 h in thisfluorescent medium. Fluo-4 is taken up by the cytoplasm of the cells.

Buffer: 145 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 10 mM Hepes, 10 mM Glucose,EGTA (Ethylene-bis oxyethylene nitrilo tetraacetic acid). The pH wasadjusted to 7.4.

Performance of the assay: A minimum of 80 μl/well of compounds offormula (I) (5×) in the above buffer (1×) were prepared(96-well-plates). The compounds of formula (I) were added to the96-well-plates at different concentrations (30 μM, 10 μM, 1 μM, 300 nM,100 nM, 10 nM, 1 nM, 100 pM, 10 pM). OT was added at a concentration of40 nM.

The relative fluorescence of Fluo-4 (λ_(ex)=488 nm, λ_(em)=590 nm) isthen measured by the FLIPR in presence or absence of compounds offormula (I). The fluorescence of the marker being sensitive to theamount of Ca²⁺, the Ca²⁺ movements can be detected. Then, it can bedetermined the ability of compounds of formula (I) to antagonize theoxytocin-induced intracellular Ca²⁺-mobilization mediated by theoxytocin receptor.

The activities of the pyrrolidine derivatives according to formula Iwere assessed using the above described in vitro biological assay.Representative values for some example compounds are given in Table II.The values refer to the concentration of the test compounds according toformula I necessary to antagonize by 50% the OT/OTR intracellularCa²⁺-mobilization. From the values, it can be derived that said examplecompounds according to formula I do exhibit a significant activity asoxytocin receptor antagonists.

TABLE II Inhibition of Ca2+ Compound mobilisation; No. IUPAC-NamehOT-RIC50 [μM] 1 (3EZ,5S)-5-(hydroxymethyl)-1- 0.03[(2′-methyl-1,1′-biphenyl-4- yl)carbonyl]pyrrolidin-3-one O-methyloxime2 (3EZ,5S)-1-(1,1′-biphenyl-4- 0.09 ylcarbonyl)-5-(hydroxymethyl)pyrrolidin-3-one O-methyloxime 4 (3Z,5S)-1-(1,1′-biphenyl-4- 0.01ylcarbonyl)-5(hydroxymethyl) pyrrolidin-3-one O-methyloxime

-   -   c) Functional Assay No. 2: Inhibition of IP3 (Inositol        Tri-Phosphate)-Synthesis in HEK/EBNA-OTR Cells

The interaction of OT on the OT-receptor leads to the IP3 synthesis,second messenger for Ca²⁺ release from sarcoplasmic reticulum, involvedin the uterine contraction triggering process (3).

This assay can be used to show the inhibition of the OT/OT-R mediatedIP3 synthesis by using test compounds of formula (I).

Stimulation of the cells: HEK/EBNA OTR (rat or human) cells are platedout into costar 12-well plates, and equilibrated for 15–24 h with 4μCi/ml radiolabelled [³H]-Inositol with 1% FCS (0.5 ml/well) and withoutinositol supplement. The medium containing the label is aspirated. DMEMmedium (without FCS, inositol), 20 mM Hepes(4-(2-hydroxyethyl)-1-piperazine-ethane-sulphonic acid), 1 mg/ml BSAcontaining 10 mM LiCl (freshly prepared), are added and incubated for10–15 min at 37° C. The agonist (i.e. oxytocin used at a concentrationof 10 nM) and the antagonists (i.e. the tests compounds of formula (I)can be used in a concentration of 10 μM, 1 μM, 300 nM, 100 nM, 10 nM, 1nM, 100 pM, 10 pM, 3 pM) can be added at the required time (15–45 min),followed by aspiration of the medium. In the presence of OT, theradiolabelled inositol is converted to radiolabelled IP3. AntagonizingOT at the OT-receptor inhibits the IP3 formation.

The amount of the radiolabelled IP3 may be determined through theensuing work-up. The reaction is stopped with 1 ml STOP-solution (i.e.0.4 M perchloric acid), and let sit for 5–10 min at Room Temperature.Then, 0.8 ml are transferred into tubes containing 0.4 ml ofneutralizing solution (0.72 M KOH/0.6M KHCO₃), and the tubes vortexedand kept in the cold at least for 2 h.

Separation of IP's: The samples are spun in a table top centrifuge at3000–4000 rpm for 15 min. 1 ml of the supernatant is transferred to newtubes containing 2.5 ml H₂O. Packed resin (Dowex AG1X8) is equilibratedwith 20 ml H₂O, and the whole samples are poured onto the chromatographycolumns, thus separating the mixture. To remove free inositol, twowashes with 10 ml H₂O are carried out.

Elution of total IP's: Elution is achieved using 3 ml 1M ammoniumformate/0.1M formic acid. The eluant is collected in scintillationcounting tubes, after the addition of 7 ml of scintillation liquid. Theamount of [³H]-IP3 is determined by a scintillating counter.

The ability of compounds of formula (I) to effectively antagonizeoxytocin-induced IP3-synthesis mediated by the oxytocin receptor, can beassessed using the above described in vitro biological assay.

-   -   d) In vivo Model for Inhibition of Uterine Contractions

The assay evaluates the biological effect of tested compounds in an invivo model of preterm labor, premature birth.

Non-pregnant Charles River CD (SD) BR female rats (9–10 weeks old,200–250 g) were treated at 18 and 24 hours before the experiment with250 μg/kg, i.p. diethylstilbestrol (DES). For the assay, the animal wasanaesthetised with urethane (1.75 g/kg, i.p.) and placed on ahomeothermic operating table. The trachea was isolated and cannulatedwith a suitable polyethylene (PE) tubing. A midline incision at thehypogastrium level was made and one uterine horn exposed, its cephalicend cannulated with a PE240 tubing and, after filling the internalcavity with 0.2 ml of sterile physiological saline, connected to a“Gemini” amplifying/recording system via a P231ID Gould Statham pressuretransducer.

One jugular vein was isolated, cannulated with a PE60 tubing andconnected to a butterfly needle to provide an i.v. route ofadministration of the test compounds via a dispensing syringe.

In the case of intraduodenal administration of the test compounds, theduodenum can be isolated and similarly cannulated through a smallincision in its wall.

One carotid artery was also isolated and cannulated with PE60 catheterand connected to a suitable syringe for blood sample collection.

After a stabilization period and throughout the experiment, the samedose of oxytocin was repeatedly injected intravenously at 30-minintervals. When reproducible contractile responses of the uterus to thesame OT stimulus (selected dose of oxytocin) were obtained, the dose ofthe test or of the reference (vehicle) was administered. Furtherinjection cycles of the same dose of oxytocin, were continued (OTinjections at 30-min intervals) for a suitable time after treatment toassess the inhibitory effects and the reversibility of these effects.

The contractile response of the uterus to oxytocin was quantified bymeasuring the intra-uterine pressure and the number of contractions. Theeffect of the reference and test compounds was evaluated by comparingpre- and post-treatment pressure values. In addition, contractions ofthe uterine were measured at 5, 40, 75, 110, 145 and 180 minutes aftertest compound administration.

The activities of the pyrrolidine derivatives claimed in the Formula Ican be assessed using the above described in vivo biological assay.Representative values for one example compound are given in Table III.The values refer to the capacity of the example compound according toFormula I to effectively antagonize oxytocin-induced uterinecontractions in the rat when administered by either intravenous or oralroute at 40 min after the test compound administration. From the valuesshown in Table III, it can be derived that said example test compoundaccording to Formula I does exhibit a significant activity as tocolytic,i.e. uterine-relaxing, agent.

TABLE III % Reduction Compound of Uterine Doses n°. IUPAC-NameContraction [mg/kg] 2 (3Z,5S)-1-(1,1′-biphenyl-4- 39.8 ± 10.0 10ylcarbonyl)-5-(hydroxymethyl) (per i.v.) pyrrolidin-3-one O-methyloxime2 (3Z,5S)-1-(1,1′-biphenyl-4- 50.9 ± 8.6 30ylcarbonyl)-5-(hydroxymethyl) (per os) pyrrolidin-3-one O-methyloxime

REFERENCES

-   1. Gimpl G. and Fahrenholz, F. Physiological Reviews 2001, 81,    629–683-   2. Maggi, M. et al. J. Clin. Endocrinol. Metabol. 1990, 70,    1142–1154.-   3. Mitchell, B. F. and Schmid, B. J. Soc. Gynecol. Invest. 2001,    8,122–33.-   4. Thorton, S. et al., Experimental Physiology 2001; 86, 297–302.-   5. Evans B. E. et al. J.Med.Chem. 1992, 35, 3919–3927.-   6. Gennaro, A. R. et al., Remington's Pharmaceutical Sciences. 18th    ed. Easton: The Mack Publishing Company, 1995.-   7. T. W. Greene et al. John Wiley & Sons Inc, Third Ed. 1999.-   8. R. C. Larock, Wiley VCH 1999.-   9. E. Breitmaier, W. Voelter Carbon-13 NMR Spectroscopy, 3rd Ed, p.    240, VCH, 1987.-   10. Philip J. Kocienski, in “Protecting Groups”, Georg Thieme Verlag    Stuttgart, New York, 1994.-   11. Cook, N. D. et al. Pharmaceutical Manufacturing International    1992; p.49–53

1. A pyrrolidine derivative of Formula I:

a geometrical isomer thereof, an optically active form thereof, anenantiomer thereof, a diastereomer thereof, one or more mixturesthereof, a racemate form thereof, or a salt thereof, wherein: R¹ isselected from the group consisting of H and C₁–C₆-alkyl; R² is selectedfrom the group consisting of hydrogen, C₁–C₆-alkyl, C₁–C₆-alkyl aryl,heteroaryl, C₁–C₆-alkyl heteroaryl, C₂–C₆-alkenyl, C₂–C₆-alkenyl aryl,C₂–C₆-alkenyl heteroaryl, C₂–C₆-alkynyl, C₂–C₆-alkynyl aryl,C₂–C₆-alkynyl heteroaryl, C₃–C₈-cycloalkyl, heterocycloalkyl,C₁–C₆-alkyl cycloalkyl, C₁–C₆-alkyl heterocycloalkyl, C₁–C₆-alkylcarboxy, acyl, C₁–C₆-alkyl acyl, C₁–C₆-alkyl acyloxy, C₁–C₆-alkylalkoxy, alkoxycarbonyl, C₁–C₆-alkyl alkoxycarbonyl, aminocarbonyl,C₁–C₆-alkyl aminocarbonyl, C₁–C₆-alkyl acylamino, C₁–C₆-alkyl ureido,amino, C₁–C₆-alkyl amino, sulfonyloxy, C₁–C₆-alkyl sulfonyloxy,sulfonyl, C₁–C₆-alkyl sulfonyl, sulfinyl, C₁–C₆-alkyl sulfinyl,C₁–C₆-alkyl sulfanyl, and C₁–C₆-alkyl sulfonylamino; R³ is selected fromthe group consisting of aryl and heteroaryl; X is selected from thegroup consisting of O and NR⁴; R⁴ is selected from the group consistingof H, C₁–C₆-alkyl, C₁–C₆-alkyl aryl, C₁–C₆-alkyl heteroaryl, aryl andheteroaryl; wherein R² and R⁴ can form together with the N atom to whichthey are linked to, a 5–8 membered saturated or unsaturatedheterocycloalkyl ring; and n is an integer from 1 to
 3. 2. A pyrrolidinederivative according to claim 1, wherein R¹ is methyl.
 3. A pyrrolidinederivative according to claim 1, wherein R³ is a phenyl.
 4. Apyrrolidine derivative according to claim 1, wherein n is an integer 1or
 2. 5. A pyrrolidine derivative according to claim 1 wherein R² and R⁴form together with the N atom to which they are linked, a 5 or 6membered cycloalkyl or heterocycloalkyl ring.
 6. A pyrrolidinederivative according to claim 1 wherein X is O or NH.
 7. A pyrrolidinederivative according to claim 1, selected from the group consisting of:(3EZ,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime;(3EZ,5S)-1-(1,1′-biphenyl-4-ylcarbonyl)-5-(hydroxymethyl)pyrrolidin-3-oneO-methyloxime;(3E,5S)-1-(1,1′-biphenyl-4-ylcarbonyl)-5-(hydroxymethyl)pyrrolidin-3-oneO-methyloxime;(3Z,5S)-1-(1,1′-biphenyl-4-ylcarbonyl)-5-[(4-methylpiperazin-1-yl)methyl]pyrro-lidin-3-oneO-methyloxime; tert-butyl{[(2S,4EZ)-1-(1,1′-biphenyl-4-ylcarbonyl)-4-(methoxyimino)pyrrolidin-2-yl]methoxy}acetate;{[(2S,4EZ)-1-(1,1′-biphenyl-4-ylcarbonyl)-4-(methoxyimino)pyrrolidin-2-yl]methoxy}aceticacid;2-{[(2S,4EZ)-1-(1,1′-biphenyl-4-ylcarbonyl)-4-(methoxyimino)pyrrolidin-2-yl]methoxy}-N-(2-pyrrolidin-1-ylethyl)acetamide;(3EZ,5S)-1-(1,1′-biphenyl-4-ylcarbonyl)-5-(methoxymethyl)pyrrolidin-3-oneO-methyloxime; (3EZ,5S)-1-(1,1′-biphenyl-4-ylcarbonyl)-5-[(4-methylpiperazin-1-yl)methyl]pyrrolidin-3-oneO-methyloxime;(3EZ,5S)-1-(1,1′-biphenyl-4-ylcarbonyl)-5-{[(4-methoxyphenyl)amino]methyl}-pyrrolidin-3-oneO-methyloxime;(3EZ,5S)-1-(1,1′-biphenyl-4-ylcarbonyl)-5-({[2-(1H-pyrazol-1-yl)ethyl]amino)methyl)-pyrrolidin-3-oneO-methyloxime;2-{[(2S,4EZ)-1-(1,1′-biphenyl-4-ylcarbonyl)-4-(methoxyimino)pyrrolidin-2-yl]methyl}-1H-isoindole-1,3(2H)-dione;(3EZ,5S)-5-(aminomethyl)-1-(1,1′-biphenyl-4-ylcarbonyl)pyrrolidin-3-oneO-methyloxime;N-{[(2S,4EZ)-1-(1,1′-biphenyl-4-ylcarbonyl)-4-(methoxyimino)pyrrolidin-2-yl]methyl}acetamide;(3EZ,5S)-1-(1,1′-biphenyl-4-ylcarbonyl)-5-(piperidin-1-ylmethyl)pyrrolidin-3-oneO-methyloxime; and(3EZ,5S)-1-(1,1′-biphenyl-4-ylcarbonyl)-5-(2-hydroxyethyl)pyrrolidin-3-oneO-methyloxime.
 8. A method of treating preterm labor, premature birth ordysmenorrhea, said method comprising administering said pyrrolidinederivative according to claim 1 to a patient in need thereof in anamount sufficient to treat said preterm labor, said premature birth orsaid dysmenorrhea.
 9. A pharmaceutical composition comprising saidpyrrolidine derivative according to claim 1 and a pharmaceuticallyacceptable carrier, diluent or excipient thereof.
 10. A process for thepreparation of said pyrrolidine derivative according to claim 1 whereinX is O, comprising O-alkylating an alcohol derivative of formula (II)with an alkylating agent R²-LG wherein LG is a leaving group

to obtain said pyrrolidine derivative.
 11. A process for the preparationof said pyrrolidine derivative according to claim 1 wherein X is NR⁴,comprising reductively aminating an aldehyde derivative of formula (XI)with an amine HNR²R⁴

to obtain said pyrrolidine derivative.